Diagnostic devices and apparatus for the controlled movement of reagents without membranes

ABSTRACT

The assay devices, assay systems and device components of this invention comprise at least two opposing surfaces disposed a capillary distance apart, at least one of which is capable of immobilizing at least one target ligand or a conjugate in an amount related to the presence or amount of target ligand in the sample from a fluid sample in a zone for controlled fluid movement to, through or away the zone. The inventive device components may be incorporated into conventional assay devices with membranes or may be used in the inventive membrane-less devices herein described and claimed. These components include flow control elements, measurement elements, time gates, elements for the elimination of pipetting steps, and generally, elements for the controlled flow, timing, delivery, incubation, separation, washing and other steps of the assay process.

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/613,650, filed on Jul. 11, 2000, (pending), which is acontinuation-in-part of U.S. patent application Ser. No. 08/828,041filed on Mar. 27, 1997, now issued U.S. Pat. No. 6,156,270, which is acontinuation in part of U.S. patent application Ser. No. 08/447,895,filed on May 23, 1995 which issued as U.S. Pat. No. 6,019,944 on Feb. 1,2000, which is a divisional applications of U.S. patent application Ser.No. 08/065,528 (abandoned), filed 19 May 1993, which was acontinuation-in-part of U.S. patent application No. 07/887,526 filed 21May 1992 which issued as U.S. Pat. No. 5,458,852, which is acontinuation in part of U.S. patent application Ser. No. 08/447,981filed on May 23, 1995, which issued as U.S. Pat. No. 5,885,527 on Mar.23, 1999, which is a divisional application of U.S. patent applicationSer. No. 08/065,528 (abandoned), filed 19 May 1993, which was acontinuation-in-pan of U.S. patent application Ser. No. 07/887,526 filed21 May 1992 which issued as U.S. Pat. No. 5,458,852 on Oct. 17, 1995;and this application is a continuation in part of U.S. patentapplication Ser. No. 08/902,775 which issued U.S. Pat. No. 6,271,040,and this application is a continuation in part of U.S. patentapplication Ser. No. 08/810,569 filed on Mar. 3, 1997 which issued asU.S. Pat. No. 6,143,576, from each of which priority is claimed, andeach of which is fully incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to devices for conducting assays, includingqualitative, semi-quantitative and quantitative determinations of one ormore analytes in a single test format.

BACKGROUND OF THE INVENTION

Over the years, numerous simplified test systems have been designed torapidly detect the presence of a target ligand of interest inbiological, environmental and industrial fluids. A synonym for targetligand is analyte or target analyte. In one of their simplest forms,these assay systems and devices usually involve the combination of atest reagent which is capable of reacting with the target ligand to givea visual response and an absorbent paper or membrane through which thetest reagents flow. Paper products, glass fibers any nylon are commonlyused for the absorbant materials of the devices. In certain cases, theportion of the absorbent member containing the test reagents is broughtinto contact, either physically or through capillarity, with the samplecontaining the target ligand. The contact may be accomplished in avariety of ways. Most commonly, an aqueous sample is allowed to traversea porous or absorbent member, such as porous polyethylene orpolypropylene or membranes by capillarity through the portion of theporous or absorbent member containing the test reagents. In other cases,the test reagents are pre-mixed outside the test device and then addedto the absorbent member of the device to ultimately generate a signal.

Commercially available diagnostic products employ a concentrating zonemethodology. In these products, such as ICON^(R) (HybritechIncorporated), TESTPACK™ (Abbott Laboratories) or ACCULEVEL^(R) (SyvaCorporation), the device contains an immunosorbing or capture zonewithin a porous member to which a member of a specific binding pair isimmobilized. The surface of the porous member also may be treated tocontain one or more elements of a signal development system. In thesedevices, there is a liquid absorbing zone which serves to draw liquidthrough the immunosorbing zone, to absorb liquid sample and reagents andto control the rate at which the liquid is drawn through theimmunosorbing zone. The liquid absorbing zone is either an additionalvolume of the porous member outside of the immunosorbing zone or anabsorbent material in capillary communication with the immunosorbingzone. Many commercially available devices and assay systems also involvea wash step in which the immunosorbing zone is washed free ofnonspecifically bound signal generator so that the presence or amount oftarget ligand in the sample can be determined by examining the porousmember for a signal at the appropriate zone.

In addition to the limitations of the assay devices and systems of theprior art, including the limitations of using absorbent membranes ascarriers for sample and reagents, assay devices generally involvenumerous steps, including critical pipetting steps which must beperformed by relatively skilled users in laboratory settings.Accordingly, there is a need for one step assay devices and systems,which, in addition to controlling the flow of reagents in the device,control the timing of the flow of reagents at specific areas in thedevice. In addition, there is a need for assay devices which do notrequire critical pipetting steps but still perform semi-quantitative andquantitative determinations.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a partially schematic, top perspective view of a device inaccordance with the present invention.

FIG. 1A is a partially schematic, perspective exploded view of thedevice showing the detail in the area of the sample addition reservoir,the sample-reaction barrier, the reaction chamber, the time gate and thebeginning of the diagnostic element.

FIG. 1B is a partially schematic, perspective exploded view of thedevice showing the detail in the area of the optional reagent reservoir,the sample addition reservoir, the sample-reaction barrier, the reactionchamber, the time gate and the beginning of the diagnostic element.

FIG. 1C is a partially schematic, perspective exploded view of thedevice showing the detail in the area of the optional reagent reservoirin fluid contact with the sample addition reservoir and the reactionchamber.

FIG. 1D is a partially schematic, perspective cutaway view of the flowcontrol means.

FIG. 2 is a partially schematic, perspective view of a second device inaccordance with this present invention, which may be used to addpre-mixed reaction mixtures.

FIGS. 3A and 3B are a partially schematic top view of the diagnosticelement showing some potential placements of capture zones.

FIG. 4 is a partially schematic, perspective view of a used reagentreservoir.

FIG. 5 is a partially schematic view of embodiments of these deviceswhich are columnar or have curved opposing surfaces.

FIGS. 6A-6F are a top view of time gates.

FIGS. 7A-7F shows typical dimensions for a preferred time gate.

FIGS. 8A-8F are a top view of sequential time gates.

FIGS. 9A-D are views of preferred textured surfaces; as illustrated atextured surface can comprise texture structures which have curved orlinear surfaces; the surfaces can be smooth or uneven. Exemplary texturestructures are conical (FIGS. 9B-C), hexagons (FIG. 9D) or mounds (FIG.9A). The structures depicted in FIG. 9 are broadly considered posts.

FIG. 10 depicts convex and concave flow fronts.

FIG. 11 depicts a preferred embodiment of a device in accordance withthe invention.

FIGS. 12A-F, respectively depict various embodiments of stops and energydirectors in accordance with the invention; FIGS. 12A, 12C and 12Edepict various embodiments without a lid attached; FIG. 12B depicts anembodiment of FIG. 12A with a lid attached, FIG. 12D depicts anembodiment of FIG. 12C with a lid attached, FIG. 12F depicts anembodiment of FIG. 12E with a lid attached. The energy directors andstops in FIG. 12 can be configured as posts or ridges.

FIG. 13 depicts a electron micrograph of an embodiment of the inventionillustrating a sample addition reservoir 1, a textured sample reactionbarrier 3, a textured reaction chamber 4, a textured used reagentreservoir 7, a stop 60, a point 70, and energy directors 62.

FIG. 14 is an enlarged view of a portion of FIG. 13, illustratingtextured sample reaction barrier 3, textured reaction chamber 4, anenergy director 62, and stop 60.

FIG. 15 depicts an electron micrograph of an embodiment of the inventionillustrating a time gate 5, a textured diagnostic lane 6, and an energydirector 62.

FIGS. 16 A-B depict electron micrographs of two views of a texturedsurface adjacent an energy director 62. The energy director depicted inthis embodiment has the form of a ridge.

SUMMARY OF THE INVENTION

The inventive devices and methods of this invention overcome theproblems found in the prior art providing devices and methods which donot require precise pipetting of sample, which do not use absorbentmembers, which include novel textures and elements for the controlledmovement of reagents in the device and which are capable of providingquantitative assays.

The devices described herein do not use bibulous or porous materials,such as membranes and the like as substrates for the immobilization ofreagents or to control the flow of the reagents through the device. Adisadvantage of, for example, membranes in diagnostic devices is that onboth microscopic and macroscopic scales the production of membranes isnot easily reproducible. This can result in diagnostic devices whichhave differential properties of non-specific binding and flowcharacteristics. Membranes are very susceptible to non-specific bindingwhich can raise the sensitivity limit of the assay. In one embodiment,the time gates of this invention can, however, be embedded in membranesor used in devices with membranes.

In the case of immunochromatographic assay formats such as thosedescribed in U.S. Pat. Nos. 4,879,215, 4,945,205 and 4,960,691, the useof membranes as the diagnostic element requires an even flow of reagentsthrough the membrane. The problem of uneven flow of assay reagents inimmunochromatographic assays has been addressed in U.S. Pat. Nos.4,756,828, 4,757,004 and 4,883,688, incorporated herein by reference.These patents teach that modifying the longitudinal edge of the bibulousmaterial controls the shape of the advancing front.

The devices of the current invention circumvent these membraneassociated problems by the use of defined surfaces, including groovedsurfaces, capillarity, time gates, novel capillary means, includingchannels and novel fluid flow control means alone or in variouscombinations, all of which are constructed from non-absorbent materials.In a preferred mode of this invention, the capillary channel of thediagnostic element is composed of grooves which are perpendicular to theflow of the assay reagents. The manufacture of grooved surfaces can beaccomplished by injection molding and can be sufficiently reproducibleto provide control of the flow of reagents through the device.

The assay devices, assay systems and device components of this inventioncan comprise two opposing surfaces disposed a capillary distance apart;at least one of the surfaces comprises the ability to detect at leastone target ligand or a conjugate in an amount related to the presence oramount of target ligand in a sample. The inventive device components maybe incorporated into conventional assay devices with membranes or may beused in the inventive membrane-less devices herein described andclaimed. Components of the invention comprise flow control elements,measurement elements, time gates, elements for the elimination ofpipetting steps, and generally, elements for the controlled flow,timing, delivery, incubation, separation, washing and other steps of theassay process.

Unlike assay devices of the prior art, the inventive assay devicesdescribed herein do not require the use of bibulous materials, such aspapers or membranes. The inventive devices of the present invention relyon the use of defined surfaces, including grooved and textured surfaces,and capillarity alone or in various combinations to move the testreagents. The inventive devices described herein provide means for thecontrolled, timed movement of reagents within the device and do notrequire precise pipetting steps. The concepts and devices of the presentinvention are especially useful in the performance of immunoassays andnucleic acid assays of environmental and industrial fluids, such aswater, and biological fluids and products, such as urine, blood, serum,plasma, spinal and amniotic fluids and the like.

Accordingly disclosed is an analytical device for determining thepresence or amount of an analyte in a test sample. The device cancomprise an array of structures, where one or more of said structureshave a surface providing an immobilized ligand receptor covalently ornon-covalently attached to said surface, and the immobilized ligandreceptor capable of binding a target ligand. The device also comprises aplurality of channels wherein said test sample containing said analyte,analyte-analog, ancillary binding member, or labeled reagent flowsthrough said channels, said analyte, analyte-analog, ancillary bindingmember, or labeled reagent diffusing across the width of said channelsand binding to said immobilized reagent.

The device can comprise an inlet port and a vent; an array ofstructures, where each structure has a surface providing an immobilizedreceptor covalently or non-covalently attached to said surface of saidstructure, and capable of binding a ligand; a plurality of channelswherein said test sample containing a ligand, flows through saidchannels, said ligand diffuses across the width of said channels to bindsaid immobilized receptor; and, a labeled reagent comprising a specificbinding member conjugated to a detectable label, where the detectablelabel is capable of producing a signal at said immobilized receptorwhich indicates the presence or amount of a ligand in a test sample.

Disclosed is an assay device comprising: a sample addition reservoir; asample reaction barrier fluidly connected to said sample additionreservoir; a reaction chamber fluidly connected to said sample reactionbarrier, said chamber having at least two fingers in the walls thereof,wherein said barrier has a higher capillarity than said reactionchamber; a time gate fluidly connected to the reaction chamber, saidtime gate capable of permitting fluid to pass therethrough at a desiredflow rate; a diagnostic element fluidly connected to the time gate, saiddiagnostic element capable of immobilizing at least one conjugate in atleast one zone; and, a used reagent reservoir fluidly connected to saiddiagnostic element, whereby fluid can flow in sequence from saidreservoir, to said barrier, to said reaction chamber, to said time gate,to said diagnostic element then to said reservoir.

Disclosed is a device capable of performing an assay, said devicecomprising two or more surfaces that are in contact by fluid duringperformance of the assay, wherein a first device surface comprises afirst immunoassay reagent immobilized thereon and a second capillaryspace surface comprises a second immunoassay reagent immobilizedthereon.

Disclosed is a device capable of performing an assay, said devicecomprising a stop and an energy director. Accordingly during manufactureof this device, the energy director serves to seal a first device pieceto a second device piece and to define a capillary space in the device,and the stop serves to allow preparation of a device chamber withuniform dimensions.

Disclosed is a zone comprising a region capable of having a fluid placedthereon, and a hydrophobic region adjacent to the region capable ofcontaining a fluid placed thereon, whereby the hydrophobic regionimpedes the flow of fluid into that hydrophobic region. Also disclosedis an assay device comprising this zone. Also disclosed is a method tofacilitate uniform drying of a liquid, where the method comprises:providing the zone; introducing liquid into the zone region capable ofhaving a fluid placed thereon; and, and drying said liquid.

Devices in accordance with the invention were used to conduct assays onliquid samples suspected of containing an analyte of interest.

Disclosed is a surface configured to facilitate placement of a uniformlayer of dried reagent thereon, said surface comprising a plurality oftexture structures, whereby a plurality of menisci are formed when afluid is placed in contact with the surface. Surfaces in accordance withthe invention, and devices comprising such surfaces, were used tofacilitate preparation of a uniform layer of a dried reagent on saidsurface.

Disclosed is a method of manufacturing analytical devices from a master.A master is provided that comprises device features in accordance withthe invention, e.g., a master having an array of structures which haveone or more channels therebetween. Thereafter, in accordance withmanufacturing techniques known to those of ordinary skill in the art,copies of the master are made.

Disclosed is a method for manufacturing a capillary space comprising ahydrophobic surface and a hydrophilic surface. The method comprisesapplying a hydrophobic material to a hydrophilic surface that is capableof forming a lumenal surface of a capillary space; or, masking a regionof a hydrophobic surface; applying a means for producing a hydrophilicsurface of the surface whereby areas of the surface which are nonmaskedbecome hydrophilic, and removing the masking to expose a hydrophobicregion of a surface that is capable of forming a lumenal surface of acapillary space.

Disclosed is a capillary space that comprises a lumen comprising atleast one rectilinear angle when viewed in a cross section, where thecapillary also comprises a hydrophobic zone on a lumenal surfacethereof. Also disclosed is a material configured to fit into a capillaryspace, said material comprising a hydrophobic zone on a surface thereof.Also disclosed is a material comprising a hydrophobic zone; where thezone, upon addition of liquid to the material, is capable of delimitinga discrete area of liquid on a surface on or within the material.

DEFINITIONS

In interpreting the claims and specification, the following terms shallhave the meanings set forth below.

Target ligand—The binding partner to one or more receptors. Synonyms fortarget ligand are analyte, ligand or target analyte.

Ligand—Binding partner to one or more ligand receptor(s). A synonym forligand is analyte. For example, a ligand can comprise an antigen, anucleotide sequence, lectin or avidin.

Ligand Analogue—A chemical derivative of the target ligand which may beattached either covalently or noncovalently to other species, forexample, to the signal development element. Ligand analogue and targetligand may be the same and both generally are capable of binding to theligand receptor. Synonyms for ligand analogue are analyte analogue ortarget analyte analogue.

Ligand Analogue Conjugate—A conjugate of a ligand analogue and a signaldevelopment element. A ligand analogue conjugate can be referred to as alabeled ligand analogue.

Signal Development Phase—The phase containing the materials involvingthe signal development element to develop signal, e.g., an enzymesubstrate solution.

Receptor—Chemical or biochemical species capable of reacting with orbinding to target ligand, typically an antibody, a binding fragment, acomplementary nucleotide sequence, carbohydrate, biotin or a chelate,but which may be a ligand if the assay is designed to detect a targetligand which is a receptor. Receptors may also include enzymes orchemical reagents that specifically react with the target ligand. Areceptor can be referred to as a reagent or a binding member. A receptorwhich is neither a labeled receptor nor an immobilized receptor can bereferred to as an ancillary receptor or an ancillary binding member. Forexample, a receptor can comprise an antibody.

Ligand Receptor Conjugate—A conjugate of a ligand receptor and a signaldevelopment element; synonyms for this term include binding memberconjugate, reagent conjugate, labeled reagent or labeled binding member.

Signal Development Element—The element which directly or indirectlycauses a visually or instrumentally detectable signal as a result of theassay process. Receptors and ligand analogues may be bound, eithercovalently or noncovalently to the signal development element to form aconjugate; when so bound these substances can be referred to as labeled.The element of the ligand analogue conjugate or the receptor conjugatewhich, in conjunction with the signal development phase, develops thedetectable signal, e.g., an enzyme.

Reaction Mixture—The mixture of sample suspected of containing targetligand and the reagents for determining the presence or amount of targetligand in the sample, for example, the ligand analogue conjugate or thereceptor conjugate. As used herein the reaction mixture may comprise aproteinaceous component which may be the target, a component of thesample or additive (e.g., serum albumin, gelatin, milk proteins).

Ligand Complement—A specialized ligand used in labeling ligand analogueconjugates, receptors, ligand analogue constructs or signal developmentelements.

Ligand Complement Receptor—A receptor for ligand complement.

Ligand Analogue-Ligand Complement Conjugate—A conjugate composed of aligand analogue, a ligand complement and a signal development element.

Capture Efficiency—The binding efficiency of the component or componentsin the reaction mixture, such as the ligand analogue conjugate or thereceptor conjugate, to the capture zone of the diagnostic element.

Capture Zone—The area on the diagnostic element which binds at least onecomponent of the reaction mixture, such as the ligand analogue conjugateor the receptor conjugate.

Capillarity—The force induced by a capillary space, or the exhibition ofcapillary action. Capillarity can be affected by the solid surface orthe liquid surface or both.

Biosensor—Any electrochemical, optical, electro-optical oracoustic/mechanical device which is used to measure the presence oramount of target ligands. For example, electrochemical biosensorsutilize potentiometric and amperometric measurements, optical biosensorsutilize absorbance, fluorescence, luminescence and evanescent waves.Acoustic/mechanical biosensors utilize piezoelectric crystal resonance,surface acoustic waves, field-effect transistors, chemical field-effecttransistors and enzyme field-effect transistors. Biosensors can alsodetect changes in the physical properties of solutions in which receptorbinding events take place. For example, biosensors may detect changes inthe degree of agglutination of latex particles upon binding antigen orthey may detect changes in the viscosity of solutions in response toreceptor binding events.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to diagnostic testing devices fordetermining the presence or amount of at least one target ligand. FIG. 1shows a preferred embodiment of a device 10 according to the invention.Generally, the devices of the invention have thicknesses of about 2 mmto 15 mm, lengths of about 3 cm to 10 cm and widths of about 1 cm to 4cm. The dimensions may be adjusted depending on the particular purposeof the assay.

One device of this invention, as depicted in FIG. 1, generallyillustrates some features of the inventive devices and portions ofdevices herein disclosed and claimed. The device 10 comprises variouselements, a sample addition zone 1, a sample addition reservoir 2, asample reaction barrier 3, a reaction chamber 4, a time gate 5, adiagnostic element 6, and a used reagent reservoir 7. The devices arecomprised of capillary channels which are formed when a top member 8 isplaced on the bottom member 9 a capillary distance apart and which movethe reagents and sample throughout the device. The top and bottommembers may be married, the various chambers sealed and the capillariesformed by a number of techniques, including but not limited to, gluing,welding by ultrasound, riveting and the like. The elements of the devicecan be used in various combinations with the diagnostic element 6 toachieve a variety of desired functions. As one skilled in the art willrecognize these elements may be combined to perform one-step ormultistep assays. The devices 10 may also be used in the formation ofreaction mixtures for the assay process. The device 20 in FIG. 2 may beused to add pre-mixed reaction mixtures for the generation of signalwhich relates to the presence or amount of the target ligand.

An optional reagent chamber 17 may be incorporated into device 10 or 20as depicted in FIG. 1B and FIG. 1C. The devices 10 and 20 may also beused with an optional fluid control means 18 as shown in FIG. 1D.

Features include, but are not limited to: 1) diagnostic elements whichare not comprised of bibulous materials, such as membranes, 2) means tocontrol the volume of sample or reaction mixture, 3) time gates, 4)novel capillary means, termed fingers herein and 5) novel flow controlmeans, sometimes referred to as a “gap” herein and 6) used reagentreservoir which prevents backward flow of reagents. Those of skill inthe art will appreciate that these elements are separately novel andnonobvious, and may be incorporated into diagnostic devices in variouscombinations and may be used with other elements known to those skilledin the art to achieve novel and nonobvious diagnostic test devices andheretofore unrealized benefits.

Components of the device (i.e., a physical structure of the devicewhether or not a discrete piece from other parts of the device) can beprepared from copolymers, blends, laminates, metallized foils,metallized films or metals. Alternatively, device components can beprepared from copolymers, blends, laminates, metallized foils,metallized films or metals deposited one of the following materials:polyolefins, polyesters, styrene containing polymers, polycarbonate,acrylic polymers, chlorine containing polymers, acetal homopolymers andcopolymers, cellulosics and their esters, cellulose nitrate, fluorinecontaining polymers, polyamides, polyimides, polymethylmethacrylates,sulfur containing polymers, polyurethanes, silicon containing polymers,glass, and ceramic materials.

Alternatively, components of the device are made with a plastic,elastomer, latex, silicon chip, or metal; the elastomer can comprisepolyethylene, polypropylene, polystyrene, polyacrylates, siliconelastomers, or latex.

Alternatively, components of the device can be prepared from latex,polystyrene latex or hydrophobic polymers; the hydrophobic polymer cancomprise polypropylene, polyethylene, or polyester.

Alternatively, components of the device can comprise TEFLON®,polystyrene, polyacrylate, or polycarbonate.

Alternatively, device components are made from plastics which arecapable of being milled or injection molded or from surfaces of copper,silver and gold films upon which are adsorbed various long chainalkanethiols. The structures of plastic which are capable of beingmilled or injection molded can comprises a polystyrene, a polycarbonate,or a polyacrylate.

Each of the elements of devices 10 and 20 will be described separately,then representative descriptions of the devices of this invention willfollow.

Sample Addition Zone

Referring to FIGS. 1 and 2, the sample addition zone 1 of the devices 10and 20 is the area where sample is introduced to the device. The sampleaddition zone 1 can be a port of various configurations, that is, round,oblong, square and the like or the zone can be a trough in the device.

Sample Addition Reservoir

Referring to FIGS. 1 and 2, the sample addition reservoir 2 is anelement of the device which receives the sample. Referring now to FIG.1, the volume of the sample addition reservoir 2 should be at least thevolume of the reaction chamber 4 or greater. The sample additionreservoir 2 can be a capillary space or it can be an open trough. Inaddition, a filter element can be placed in or on the sample additionreservoir 2 to filter particulates from the sample or to filter bloodcells from blood so that plasma can further travel through the device.The sample addition reservoir can comprise a vent (not illustrated) tofacilitate escape of gas and liquid filling of the reservoir.

In a preferred embodiment, the volume or capacity of the sample additionreservoir 2 is 1 to 5 times the volume of the reaction chamber 4. Ingeneral, one selects a volume or capacity of this reservoir 2 such thatif the excess sample is used to wash the diagnostic element 6 thenenough volume of sample is needed to thoroughly remove any unboundreagents from the diagnostic element 6 arising from the assay process.

Reservoir 2 may also contain certain dried reagents which are used inthe assay process. For example, a surfactant can be dried in thisreservoir 2 which dissolves when sample is added. The surfactant in thesample would aid in the movement of the sample and reaction mixturethrough the device by lowering the surface tension of the liquid. Thesample addition reservoir 2 is generally in direct fluid contact withthe sample-reaction barrier 3 (FIG. 1) or the diagnostic element 6 (FIG.2).

Sample-Reaction Barrier

As depicted in FIG. 1, the sample-reaction barrier 3 separates thesample in the sample addition reservoir 2 from the reaction mixture inthe reaction chamber 4. The sample-reaction barrier is desired becauseit provides the device with the capability of forming a precise reactionmixture volume. A precise volume of the reaction mixture is generallynecessary for assays in which semi-quantitative or quantitative resultsare desired. Thus, a precise pipetting step of the sample to the deviceis not required because the sample reaction barrier forms a reactionchamber of precise volume into which the sample is capable of flowing.The sample reaction barrier 3 is desired because the reactions whichtake place in the reaction chamber 4 should preferably be separated fromthe excess sample in the sample addition reservoir 2.

The sample reaction barrier 3 comprises a narrow capillary, generallyranging from about 0.01 mm to 0.2 mm and the surfaces of the capillarycan be smooth or have a single groove or a series of grooves which areparallel or perpendicular to the flow of sample. In a preferredembodiment of the sample reaction barrier 3, now referring to FIG. 1A,grooves 12, parallel to the flow of sample, are incorporated onto onesurface of the device a capillary distance, for example, 0.02 mm to 0.1mm, from the other surface. The volume of sample which fills thesample-reaction barrier 3 (FIG. 1A) should be kept to a minimum, fromabout 0.01% to 10% of the reaction chamber 4 volume so that the reagentsof the reaction chamber 4 do not significantly diffuse back into thesample in the sample addition reservoir 2. That is, the diffusion of thereaction mixture back into the excess sample should be kept to a minimumso that the chemical or biochemical reactions occurring in the reactionmixture are not substantially influenced by the excess sample in thesample addition reservoir 2. Groove depths can range from about 0.01 mmto 0.5 mm and preferably from about 0.05 mm to 0.2 mm. When more thanone groove is used for this element, the number of grooves in thiselement is typically between 10 and 500 grooves per cm and preferablyfrom about 20 to 200 grooves per cm. Sample from the sample additionreservoir 2 flows over the grooves 12 by capillary action and then intothe reaction chamber 4. In a further preferred embodiment, grooves,hereafter termed “fingers” 16, are situated in the wall of the reactionchamber 4 in fluid contact with the grooves 12 or capillary space of thesample-reaction barrier 3. These fingers 16 are typically 0.5 mm to 2 mmwide, preferably 1 mm to 1.5 mm wide and typically 0.1 mm to 1.5 mm indepth, preferably about 0.2 to 1 mm in depth. The fingers 16 in the wallof the reaction chamber 4 aid in the capillary flow of the sample intothe reaction chamber 4. That is, the fingers allow fluid to move from acapillary where the capillarity is relatively high to a capillary wherethe capillarity is lower. Thus, the capillary at the sample-reactionbarrier is generally more narrow and has a greater capillarity than thecapillary or space of the reaction chamber. This difference incapillarity can cause the flow of sample or fluid in the device to stopin the sample-reaction barrier capillary. Presumably, the fingers breakthe surface tension of the fluid at the interface of the two capillariesor spaces and thereby cause the fluid to move into a capillary or spaceof lower capillarity. One can appreciate that the utility of fingers canbe extended to any part of the device where fluid must flow from highcapillarity to low capillarity. In practice, this is usually when thedirection of fluid flow is from a narrow capillary (highercapillarity)to a wider capillary (lower capillarity).

The top surface of the sample reaction barrier may also be used toimmobilize reagents used in the assay process such that the sample flowsover the sample reaction barrier, dissolves the reagents and moves intothe reaction chamber. The movement of the sample and reagents into thereaction chamber may act as a mixing means.

Reaction Chamber

Referring to FIG. 1, the sample moves into the reaction chamber 4 fromthe sample-reaction barrier 3. The reagents of the device 10 arepreferably placed in the reaction chamber 4, for example, as dried orlyophilized powders, such that when the sample enters the reactionchamber 4 the reagents quickly reconstitute. The volume of the reactionchamber 4 is the volume of sample which defines the reaction mixture.The reaction chamber may be sealed on two sides, for example, byultrasonic welding of the top and bottom members. Thus, delivery of thesample to the device 10 at the sample addition zone 1 does not require aprecise pipetting step to define the volume of the reaction mixture.Mixing features which mix the reaction mixture can also be incorporatedin conjunction with the reaction chamber element 4, such as thosedescribed in U.S. Patent application Ser. No. 711,621 filed Jun. 5,1991, now abandoned, hereby incorporated by reference. The sample fillsthe reaction chamber 4 because of capillary forces and also,potentially, because of the hydrostatic pressure exerted by the samplein the sample addition reservoir 2.

A surface of reaction chamber 4 may be smooth or comprised of texturestructures such as posts or grooves. Texture on a device surface canfacilitate drying of reagents on the surface during preparation of thedevice, and can facilitate movement of sample into the reaction chamber4. Texture on a device surface facilitates uniform placement of driedreagents on the surface as follows: A liquid reagent-containing fluid isplaced in contact with the textured surface, and small reagent fluidmenisci form adjacent each texture structure. Absent the presence oftexture, the fluid would tend to form larger menisci at corners of theentire chamber, which when dried would produce a non-uniform layer ofdried reagent. When texture structures are designed into the device, thepresence of numerous small menisci leads to a more uniform layer ofreagent that is dried throughout the chamber.

The volume of the reaction chamber 4, and thereby the reaction mixture,may be any volume which accommodates the reagents and which provides thedesired sensitivity of the assay. The shape of the reaction chamber 4should be such that the movement of the reaction mixture from thereaction chamber 4 is not turbulent and eddies are not formed as aresult of the movement out of the reaction chamber 4. A preferred shapeof the reaction chamber 4 is shown in FIG. 1. The depth of the reactionchamber 4 should be commensurate with the width of the chamber toaccommodate the desired reaction mixture volume. The depth of thereaction chamber can range from about 0.05 mm to 10 mm and preferablyfrom 0.1 mm to 0.6 mm. To accommodate a particular volume of thereaction chamber, the length and width of the reaction chamber should beadjusted and the depth maintained as narrow as is practical. Thereaction chamber 4 is in direct fluid contact with the sample-reactionbarrier 3 and the diagnostic element 6 or time gate 5. In addition, thereaction chamber 4 may also be in direct fluid contact with an optionalreagent reservoir 17 as shown in FIGS. 1B and 1C.

A preferred embodiment of the reaction chamber utilizes a ramp whichextends from the bottom of the reaction chamber to the surface of thediagnostic element. The ramp minimizes or prevents mixing and eddyformation of the reaction mixture with the sample at the interface ofthe reaction chamber and the diagnostic element as the fluid movesthrough the device. Thus, the ramp allows a smooth transition of thefluid out of the reaction chamber and onto the diagnostic element. Thelength of the ramp should be optimized for each depth of the reactionchamber, but generally, the ramp is at an angle of between 25 and 45degrees relative to the floor of the reaction chamber.

Time Gate

Referring to FIG. 1A, the time gate 5 holds the reaction mixture in thereaction chamber 4 for a given period of time. The concept of the timegate is that a predominantly aqueous solution cannot pass through asufficiently hydrophobic zone until the hydrophobic zone is madesufficiently hydrophilic. Furthermore, the hydrophobic zone is madehydrophilic through the binding of a component in the aqueous solutionto the hydrophobic zone. The sufficiently hydrophobic zone is generallyin a capillary space. The driving force for fluid movement over orthrough the time gate may be either the capillarity of the space orhydrostatic pressure exerted by the sample or a combination of both ofthese forces. The amount of time which is required to hold the reactionmixture in the reaction chamber 4 is relative to the assay process suchthat the reactions which occur in the reaction chamber 4 as a result ofthe assay process will reflect the presence or amount of target ligandin the sample. Thus, the time gate 5 delays the flow of the reactionmixture onto the diagnostic element 6. The time gate 5 delays the flowof the reaction mixture by the principle that a hydrophilic liquid, suchas an aqueous solution or one which has a dielectric constant of atleast 40, cannot move past a sufficiently hydrophobic barrier in acapillary channel. In designing and building a time gate, one can beginwith a hydrophobic surface, such as are found on native plastics andelastomers (polyethylene, polypropylene, polystyrene, polyacrylates,silicon elastomers and the like) or silicon chip surfaces or metalsurfaces, either smooth, grooved or textured and a capillary is formedby an opposing surface which can be hydrophobic or hydrophilic in natureand smooth, grooved or textured. The hydrophobic surface(s) in thecapillary have a microscopic surface area onto which can bind componentswhich are generally soluble in a predominantly aqueous solution. Thehydrophilic character and the concentration of the component(s) in thereaction mixture and the overall surface area of the time gate affectsthe mechanics of the time gate. The amount of time for which the timegate 5 holds the reaction mixture is related to the rate of binding of acomponent(s) from the reaction mixture to the hydrophobic barrier. Thebinding of the component(s) from the reaction mixture changes thehydrophobic barrier to a zone which is sufficiently hydrophilic overwhich or through which the reaction mixture can flow. Creating thesufficiently hydrophilic surface then allows the fluid to flow as if thetime gate had not been in the device. Thus, fluid flow through theremainder of the device is not affected once the time gate has been madehydrophilic. Other devices described which incorporate fluid delaymeans, for example, in U.S Pat. Nos. 4,426,451 and 4,963,498, herebyincorporated by reference, only require an external manipulation of thedevice to start fluid flow or utilize capillary constrictions to slowfluid flow. In this latter case, the capillary constriction used todelay fluid flow will affect the fluid flow through the remainder of thedevice.

In a preferred embodiment, for example, the time gate 5 can be composedof latex particles 15 (FIG. 1A, not drawn to scale), such as polystyrenelatexes with diameters of between about 0.01 μm and 10 μm or hydrophobicpolymers, such as polypropylene, polyethylene, polyesters and the like,which are introduced onto the device in the appropriate zone where thereaction mixture must travel. In another preferred embodiment, the timegate can be created by application of a hydrophobic chemical, such as anink or a long chain fatty acid, or a hydrophobic decal to the desiredzone. The hydrophobic chemical or decal is generally not soluble or ispoorly soluble in the reaction mixture. In yet another preferredembodiment, the time gate can also be formed by changing a hydrophilicsurface to a hydrophobic surface. For example, hydrophobic surfaces madehydrophilic by plasma treatment can be converted back to a hydrophobicsurface by the application of solvents, ultraviolet light or heat andthe like. These treatments can act to change the molecular structure ofthe hydrophilic, plasma modified surface back to a hydrophobic form.

The component(s) in the reaction mixture which bind to the hydrophobiczone may be various proteins, polypeptides, polymers or detergents. Apreferred protein is bovine serum albumin. The time delay provided bythe time gate 5 depends on the concentration of the component(s) in thereaction mixture, for example, bovine serum albumin, which binds to thehydrophobic zone, for example, the surface area provided by the latexparticles 15. Another preferred embodiment of the time gate 5 utilizespolyelectrolytes which are hydrophobic and which become hydrophilic byexposure to the buffering capacity of the reaction mixture. The timegate 5 would be comprised of, for example, polyacrylic acid, which inits protonated form it is hydrophobic. The reaction mixture, if bufferedabove the pK_(a) of the polyacrylic acid, would deprotonate the acidgroups and form the hydrophilic salt of the polymer. In this case, thetime delay is related to the mass of polyelectrolyte and the pH and thebuffering capacity of the reaction mixture.

The geometry or shape of the time gate can influence the area of thetime gate that the fluid will pass over or through. That is, the timegate can be designed to direct the flow of liquid through a specificarea of the time gate. By directing the fluid to flow through a definedarea of the time gate the reproducibility of the time delay is improved.FIG. 6 shows representative geometries of time gates. For example, asshown in FIG. 6, time gates a-d, the time gates have V-shapesincorporated into their design, and more specifically, the length of thetime gate (defined as the distance the fluid must cross over or throughin order to pass the time gate) is less at the tip of the V than in thebody of the time gate. Thus, in a preferred mode, the fluid will crossover or pass through the time gate where the length is shortest therebydirecting fluid flow through the time gate in a consistent manner. Ingeneral, the directionality of fluid flow over or through the time gatesis represented by opposing arrows in FIG. 6. In a preferred embodiment,the orientation of the time gates b, c and d of FIG. 6 are such that thefluid touches the flat portion of the time gate first rather than the Vshape. In other words, the preferred direction of flow for the timegates b, c and d of FIG. 6 is represented by the up arrow. In caseswhere the time gate is simply a line, for example as seen in FIG. 6,time gate e and f, the path of fluid flow over or through the time gatecan occur at any point on the time gate. Thus, the time gates which havegeometries directing the fluid flow over or through a consistent area ofthe time gate are preferred. For example, time gates with lengthsranging from about 1.3 mm to 0.13 mm achieve delay times ofapproximately 0.3 min to 5.5 min, respectively, when the distancebetween surfaces is about 0.018 mm. When the time gate is V-shaped, thelength of the time gate 4 at the tip of the V has dimensions smallerthan the length of the time gate at the remaining portion of the V; thatis, the arms of the V should have a length roughly 2 to 5 times thelength of the V tip, as for example, FIG. 7, time gate a, illustrates.FIG. 7, time gate b, shows that only a small area of the time gate iscrossed over or through at the tip of the V as compared with theremainder of the time gate. The time gate should span the width of thecapillary or space so that the entire fluid front comes in contact withthe time gate. If the time gate was not as wide as, for example, thediagnostic element, then the fluid front would go around the time gate.Thus, the time gate should “seal” the fluid in the space during thedelay period.

Referring to FIG. 1, one skilled in the art can recognize that eachdevice 10 could incorporate one or more time gates to achieve thedesired function of the device. FIG. 8 shows some examples of thesequential placement of several time gates of FIG. 6. For example, asdiscussed in the next section, Optional Reagent Chambers, if asequential addition immunoassay was to be performed by the device thentwo time gates would allow two sequential incubation steps to beperformed by the device prior to the movement of the reaction mixture tothe diagnostic element. In another example, if an incubation of thereaction mixture on the capture zone or zones of the diagnosticelement(s) 6 was required then a time gate(s) would be placedimmediately behind the capture zone or zones. This use of the time gatemay arise in cases where poor efficiency of binding of the component inthe reaction mixture to the capture zone of the diagnostic element wouldprevail.

Another application of the time gate involves the placement of a timegate on a surface which is not part of a capillary space. For example,the time gate can be placed on a hydrophilic surface, which alonewithout a capillary space will allow liquids to move. This is generallythe case when a substantial volume of liquid is placed on a surface andit spreads because of surface tension and because of the hydrostaticpressure of the liquid pushing the meniscus outwardly. The time gatethen would function to delay the advance of the fluid front because thehydrostatic nature of the surface of the time gate would stop themovement of liquid. As the meniscus of the advancing liquid touches thetime gate, the component or components in the liquid binds to the timegate to create a sufficiently hydrophilic surface for a continuedadvance of the liquid on the surface.

Yet another embodiment of the time gate involves the positioning of atime gate prior to a membrane which is used to capture a conjugate orreceptor. In yet another embodiment of the time gate, the time gate canbe composed of hydrophobic surfaces in a membrane. In those cases, thehydrophobic membrane is positioned prior to the portion of membranewhich captures the conjugate or receptor and may be positioned after areaction chamber or a portion of membrane where reagents of the assayare placed or embedded and where the reagents incubate for a definedperiod of time. The time gate in the membrane can be formed byapplication of raw latex particles in the membrane at an appropriatesolids concentration ranging from about 0.01% to 10%. The size of thelatex particles should be slightly less than the pore size of themembrane so that the latex becomes imbedded within the membrane. Thedensity of latex within the membrane at the time gate should be uniformso that the reaction mixture does not circumvent the time gate. Forexample, the latex size used to create a time gate for a membrane with apore size of 1 μm can range between 0.05 and 0.2 μm. Since thedistribution of pore sizes in membranes varies widely, the actual sizeof latex used must be arrived at by experimentation. The hydrophobicnature of the membrane used for the time gate can also be formed byplasma treatment or by treatment of the membrane with hydrophobicchemicals or polymers that adsorb to the membrane. One skilled in theart can appreciate that the teachings described herein of the inventivefeatures of the time gate can be utilized to design time gates in avariety of diagnostic devices which utilize membranes. That is, devicesdescribed, for example, in U.S. Pat. Nos. 4,435,504, 4,727,019,4,857,453, 4,877,586 and 4,916,056, hereby incorporated by reference,can incorporate a time gate, for example, prior to the membrane or inthe membrane which captures the conjugate or receptor.

Optional Reagent Chambers

Referring to FIGS. 1B and 1C, the optional reagent chamber 17 is usefulfor the introduction of reagents into the assay process. In general, theoptional reagent chamber 17 may be in direct fluid contact with thesample addition reservoir 2 via a sample reaction barrier 3 or a portthe reaction chamber 4 or the diagnostic element 6, via a samplereaction barrier 3 or a port. For example, FIG. 1B shows the optionalreagent chamber 17 in direct fluid contact with the reaction chamber 4.The flow of the introduced reagent may be controlled by a time gate 5 aand fingers 16 can aid in the movement of reagents into the reactionchamber 4. Referring now to FIG. 1C, for example, if a sequentialaddition immunoassay was to be performed by the device then 2 time gates5 and 5 a would and fingers 16 can aid in the movement of reagents intothe reaction chamber 4. Referring now to FIG. 1C, for example, if asequential addition immunoassay was to be performed by the device then 2time gates 5 and 5 a would allow 2 sequential incubation steps to beperformed in the optional reagent chamber 17 and then in the reactionchamber 4 by the device prior to the movement of the reaction mixtureonto the diagnostic element 6. That is, sample would be applied to thesample addition reservoir 2 through the sample addition zone 1 and thesample flows over the sample reaction barrier 3 and into the optionalreagent chamber 17 by the aid of fingers 16 where the first set ofreactions would occur. The time gate 5 a, after the appropriate amountof time, would allow the reagents to flow over the sample reactionbarrier 3 a and into the reaction chamber 4 by the aid of fingers 16 awhere the next set of reactions would take place. After the appropriateamount of time, the time gate 5 allows the flow of reaction mixture ontothe diagnostic element 6.

Fluid Control Means

Referring to FIG. 1D, the optional fluid control means 18 is designed tocontrol the flow of the reaction mixture in the device. Morespecifically, the optional fluid control means 18 causes the volume ofthe reaction mixture to flow over the capture zone of the diagnosticelement 6 at a rate which allows for an optimum capture of reagents ontothe capture zone. After the volume of the reaction mixture flows overthe capture zone the rate of flow of the excess reagents may beincreased. The differential rate of flow of the reagents in the deviceis achieved by designing a gap 18 between the surfaces of the capillaryspace 19 of the diagnostic element 6. The size of the gap 18 is largerthan the capillary space 19 of the diagnostic element 6. The gap 18generally follows the capture zone or the zone where the rate of flow isrequired to be decreased. The gap 18 in the diagnostic element 6 thushas an associated volume. The volume of the gap 18 is filled with thereaction mixture by capillary action as it moves through the device.Since the gap 18 after the capture zone is greater than the capillaryspace 19 of the diagnostic element 6 a drop in capillary pressure at thebeginning of the gap 18 results in a decrease in the rate of flow of thereaction mixture into the gap 18 and therefore a decrease in the rate offlow of the reaction mixture over the capture zone. Varying the size ofthe gap 18 changes the capillarity in the gap and thus the flow of thereaction mixture over the capture zone. In the case of immunoassaysrequiring a wash step to remove unbound reagents from the diagnosticelement 6, it is generally desired that the rate of flow of the washsolution over the diagnostic element 6 is faster than the rate of flowof the reaction mixture over the diagnostic element 6 because thisdecreases the time of the assay. The shape of the gap can take manyforms. As shown in FIG. 1D, the gap has square corners, however, the gapcan be shaped as a trapezoid or triangle which would change the rate offlow of the reaction mixture while flowing into the gap. One skilled inthe art can also appreciate that for certain immunoassays a wash step isnot required.

The control of the rate of flow of the reagents in the device can alsobe used to allow chemical reactions to take place in one zone of thedevice before the reagents move to another area of the device where theextent of reaction of the reagents is monitored or where furtherreaction may take place. For example, several fluid control means couldbe incorporated into a device for use in immunoassays where a sequentialaddition and incubation of reagents is necessary. That is, the samplecomes in contact with the first reagents and the time for the reactionof the sample and first reagents is controlled by a first gap. When thefirst gap is filled with fluid, the reaction mixture continues to thesecond reagents at which time an additional chemical reaction cansubsequently take place. The time required for completion of this secondreaction can also be controlled by a second gap before further flow ofthe reaction mixture along the diagnostic element. Chemical andbiochemical reactions also take place in the volume of the gap, forexample, by immobilizing reagents in the gap.

Diagnostic Element

Referring to FIGS. 1 and 2, the diagnostic element 6 is formed byopposing surfaces which are a capillary distance apart through which thereaction mixture flows and on which are placed one or more capturezones. The capture zones are comprised of reagents, such as receptors,or devices, such as biosensors which bind or react with one or morecomponents from the reaction mixture. The binding of the reagents fromthe reaction mixture to the capture zones of the diagnostic element 6 isrelated to the presence or amount of target ligand in the sample. One ormore receptors or biosensors can be placed on the diagnostic element 6to measure the presence or amount of one or more target ligands. Thereceptors or biosensors can be placed in discrete zones on thediagnostic element 6 or they can be distributed homogeneously orheterogeneously over the surface. Receptors or other chemical reagents,for example, a receptor against the signal generator can also beimmobilized on the diagnostic element 6 to verify to the user that thereagents of the reaction mixture are viable and that the reactionmixture passed through the zones of the receptors or biosensors. Asingle receptor or biosensor can be placed over the majority of thediagnostic element 6 such that as the reaction mixture flows through thediagnostic element 6 the components from the reaction mixture bind tothe surface of the diagnostic element 6 in a chromatographic fashion.Thus, the distance which the component of the reaction mixture bindswould be related to the concentration of the target ligand in thesample. The reagents, such as receptors, are immobilized on the surfaceof the diagnostic element 6 through covalent bonds or throughadsorption. A preferred embodiment is to immobilize receptor coatedlatex particles, for example of diameters ranging from about 0.1 μm to 5μm. In addition, particles termed “nanoparticles” can also be coatedwith receptor and the resulting nanoparticles can be immobilized to thediagnostic element through adsorption or covalent bonds. Nanoparticlesare generally composed of silica, zirconia, alumina, titania, ceria,metal sols, and polystyrene and the like and the particle sizes rangefrom about 1 nm to 100 nm. The benefit of using nanoparticles is thatthe surface area of the protein coating the nanoparticle as a functionof the solids content is dramatically enhanced relative to larger latexparticles. The surfaces of the diagnostic element 6 would allow thereceptor coated nanoparticles or latex particles to bind to thediagnostic element 6. In a preferred embodiment, the receptors bind tothe surface of the diagnostic element through electrostatic, hydrogenbonding and/or hydrophobic interactions. Electrostatic, hydrogen bondingand hydrophobic interactions are discussed, for example, in Biochemistry20, 3096 (1981) and Biochemistry 29, 7133 (1990). For example, thediagnostic element 6 can be treated with a plasma to generate carboxylicacid groups on the surface. The receptor coated latex particles arepreferably applied to the diagnostic element 6 in a low salt solution,for example, 1-20 mM, and at a pH which is below the isoelectric pointof the receptor. Thus, the negative character of the carboxylic acidgroups on the diagnostic element 6 and the positive charge character ofthe receptor latex will result in enhanced electrostatic stabilizationof the latex on the diagnostic element 6. Hydrogen bonding andhydrophobic interactions would also presumably contribute to thestabilization and binding of the receptor latex to the diagnosticelement 6. Magnetic fields may also be used to immobilize particleswhich are attracted by the magnetic field.

In an additional embodiment of the diagnostic element, now referring toFIG. 5, the diagnostic element 6 is a cylindrical surface which may becomposed of grooves. When the diagnostic element is composed of grooves,the grooves generally run perpendicular to the flow of the reactionmixture. A capillary space is formed around the diagnostic element by around tube which is generally clear; thus, the surface of the diagnosticelement and the opposing surface of the tube are a capillary distanceapart. The capillary formed allows the flow of the reaction mixture overthe round diagnostic element 6. Generally, the reaction mixture wouldtravel up against gravity or down with gravity through the cylindricalcapillary space. The capture zones of the round diagnostic element 6 canbe placed in discrete zones or over the entire length of the diagnosticelement 6. The capture zones may also circle the diameter of thediagnostic element 6 or may be applied to only a radius of thediagnostic element 6. The reaction mixture may be delivered to thediagnostic element 6 through the tube 8. Furthermore, the cylindricalvolume of the tube 8 may be used as a reaction chamber 4 and a discshaped sample reaction barrier 3 with grooves on its perimeter may alsobe inserted to form the reaction chamber 4 and the sample additionreservoir 2. From this discussion, now referring to FIGS. 1 and 2, oneskilled in the art can also appreciate that the flat diagnostic element6 may also be curved such that the curvature is a radius of a circle.

One skilled in the art can appreciate that various means can be used forthe detection of signal at the capture zone of the diagnostic element.In the case of the use of biosensors, such as, for example, apiezoelectric crystal, the piezoelectric crystal onto which would beimmobilized a receptor, would be the capture zone and the responsegenerated by binding target ligand would be generally reflected by anelectrical signal. Other types of detection means include, but are notlimited to visual and instrumental means, such as spectrophotometric andreflectance methods. The inventive features of the diagnostic elementdescribed herein allows for improved capture efficiencies on surfacesover which a reaction mixture flows and that various means for detectionmay be used by one skilled in the art. The surfaces of the capillariesin the device are generally hydrophilic to allow flow of the sample andreaction mixture through the device. In a preferred embodiment thesurface opposing the diagnostic element 6 is hydrophobic such that thereaction mixture repels this surface. The repulsion of reaction mixtureto the surface opposing the diagnostic element 6 forces the reactionmixture, and particularly the protein conjugates, to the surface wherecapture occurs, thus improving the capture efficiency of the componentsof the reaction mixture to the capture zone. The hydrophobic surfacesopposing the diagnostic element can have a tendency to becomehydrophilic as the reaction mixture progresses through the diagnosticelement because various components which may be present endogenously orexogenously in the sample or reaction mixture, such as, for example,proteins or polymers, bind to the hydrophobic surface. A preferredhydrophobic surface opposing the diagnostic element can be composed ofTEFLON®. It is well known to those skilled in the art that TEFLON®surfaces bind proteins poorly. Thus, the TEFLON® surface opposing thediagnostic element would not become as hydrophilic as would surfacescomposed of, for example, polystyrene, polyacrylate, polycarbonate andthe like, when the reaction mixture flows through the diagnosticelement.

In another preferred embodiment, the diagnostic element 6 is hydrophilicbut the areas adjacent to the diagnostic element 6 are hydrophobic, suchthat the reagents of the assay are directed through only the hydrophilicregions of the diagnostic element. One skilled in the art will recognizethat various techniques may be used to define a hydrophilic diagnosticelement or zone, such as plasma treatment of hydrophobic surfaces usingmasks which shield the surfaces, except for the diagnostic element, fromthe treatment or by application of hydrophobic adhesives to hydrophilicsurfaces to define a diagnostic element or by the use of viscoushydrophobic compounds, such as an, oil or a grease. In another preferredembodiment, the capillary of the diagnostic element can be formed byultrasonic welding. The boundaries of the diagnostic element aredictated by the energy directors which are used to form the sonicatedweld.

The surfaces of the diagnostic element 6 or of the other components ofthe device may be smooth, grooved, or grooved and smooth. Varioustextured surfaces may also be employed, alone or in combination withsmooth or grooved surfaces. For example, surfaces composed of posts,grooves, pyramids, and the like referred to as protrusions; or holes,slots, waffled patterns and the like, referred to as depressions may beutilized. Referring now to FIG. 9, the surface can comprise texturestructures that comprise the form of diamonds, hexagons, octagons,rectangles, squares, circles, semicircles, triangles or ellipses. Thetextured surface can comprise texture structures in geometries orderedin rows, staggered or totally random; different geometries can becombined to yield the desired surface characteristics. Typically, thedepressions or protrusions of the textured surface can range from about1 nm to 0.5 mm and preferably from about 10 nm to 0.3 mm; the distancebetween the various depressions or protrusions can range from about 1 nmto 0.5 mm, and preferably from about 2 nm to 0.3 mm.

A surface of diagnostic element 6 may be smooth or comprised of texturestructures such as posts or grooves. Texture on a device surface canfacilitate drying of reagents on the surface during preparation of thedevice, and can facilitate movement of sample in the diagnostic element.Texture on a device surface facilitates uniform placement of driedreagents on the surface as follows: A liquid reagent-containing fluid isplaced in contact with the textured surface, and small reagent fluidmenisci form adjacent each texture structure. Absent the presence oftexture, the fluid would tend to form larger menisci at corners of theentire surface or chamber, which when dried would cause a non-uniformlayer of dried reagent. When texture structures are designed into thedevice, the presence of numerous small menisci leads to a more uniformlayer of reagent that is dried throughout the surface or chamber.

In a preferred mode as shown in FIGS. 1 and 2, one surface of thediagnostic element 6 is grooved and the grooves are perpendicular to theflow of the reaction mixture and the opposing surface is smooth. Inanother embodiment, one surface of the diagnostic element 6 is groovedat the capture zone and the areas adjacent to the capture zone aresmooth. The opposing surface of the diagnostic element 6 may be smoothor may be grooved, for example, the grooves of each surface intermesh.The positioning of the grooves of the diagnostic element perpendicularto the flow of the reaction mixture is beneficial in that the flow ofthe reaction mixture through the diagnostic element 6 occurs in anorganized manner with a distinct, straight front dictated by the groovesin the capillary space.

In addition, when one surface is in close proximity, for example 1 μm to100 μm, to the peaks of the grooves then the capture efficiency of thecomponents from the reaction mixture can be enhanced. The enhancement ofcapture efficiency at the capture zones in grooved diagnostic elementsas compared to smooth surface elements may be related to the movement ofthe reaction mixture in the capillary space; that is, in the case of thegrooved surface the reaction mixture is forced to move over the peak ofthe groove and into the trough of the next groove. Thus, a finer groovedsurface, that is, more grooves per cm, would provide a better captureefficiency than a coarser grooved surface. The reaction mixture is thusdriven closer to the surface of the grooved diagnostic element than itwould be if both surfaces were smooth.

Also, the close proximity of the surfaces decreases the volume of thebulk reaction mixture above the surface of the diagnostic element andtherefore decreases the diffusion distance of the components which bindto the diagnostic element. The proximity of the surfaces of thediagnostic element should minimize the volume of reaction mixture in thediagnostic element at the capture zone without blocking the capillaryflow through the element. In addition, in embodiments where a reagent isdried on a device surface and another reagent is dried on a separatesurface of the device, these reagents can diffuse from their respectivesurfaces upon introduction of fluid to those surfaces. The surfaceshaving reagent immobilized thereon can be surfaces in a particularchamber of the device or can be surfaces in different regions of thedevice. The regions can be separate chambers or can be device surfacesthat do not delimit a chamber.

The capture of, for example, the complex of target ligand: Ligandreceptor conjugate at the capture zone can approach 100% efficiency ifthe proximity of the surfaces is optimized. The capture of nearly all ofthe ligand receptor conjugate which is bound by target ligand is mostdesired because a greater sensitivity of the assay as a function ofsample volume can be achieved. Other advantages of improved captureefficiency are that less reagents are used because the sample volume isdecreased, the assay device can be miniaturized because of the smallersample volume and the reproducibility of the assay result will beimproved because changes in the rate of flow of the reaction mixturethrough the capture zones will have less or no effect on the capture ofthe labeled conjugates.

The capillary space can be defined by a variety of ways, for example,machining the surfaces to the appropriate tolerances or using shimsbetween the surfaces. In a preferred embodiment, ultrasonic welding ofthe surfaces defines the capillary. In this case, the capillary space isdefined by the energy directors and the distance between the surfaces isa function of the size of the energy director, the welding energy, thetime of energy application and the pressure applied during welding. Thesurfaces of the diagnostic element can be parallel or non-parallel. Inthe latter case, the flow rate of the reagents through the diagnosticelement will not be uniform throughout the length. A preferredembodiment is to maintain the surfaces of the diagnostic elementapproximately parallel. The surfaces of the diagnostic element can bemade from materials, such as plastics which are capable of being milledor injection molded, for example, polystyrene, polycarbonate,polyacrylate and the like or from surfaces of copper, silver and goldfilms upon which are adsorbed various long chain alkanethiols asdescribed in J. Am. Chem. Soc. 1992, 114, 1990-1995 and the referencestherein. In this latter example, the thiol groups which are orientedoutward can be used to covalently immobilize proteins, receptors orvarious molecules or biomolecules which have attached maleimide or alkylhalide groups and which are used to bind components from the reactionmixture for determining the presence or amount of the target ligand.

Referring to FIGS. 3A and 3B, the zones of immobilization of one or morereceptors or the placement of biosensors at the capture zone 17 on thediagnostic element 6 can take many forms. For example, if the targetligand is very low in concentration in the sample then one would desirethat all of the reaction mixture pass over the zone of immobilizedreceptor or biosensor to obtain the best signal from the given volume ofreaction mixture. In this case, the placement of the reagents orbiosensors on the diagnostic element 6 at the capture zones 17 could,for example, resemble that shown in FIG. 3A. If the target ligand in thesample is high in concentration and the sensitivity of the analyticalmethod is not an issue then the placement of the receptors or biosensorsat the capture zones 17 could, for example, resemble that in FIG. 3B.One skilled in the art can appreciate that the placement of receptors orbiosensors on the diagnostic element is a function of the sensitivityrequirements of the analytical method.

One or more diagnostic elements can be comprised in a device. Thereaction mixture may be applied to a device with multiple diagnosticelements. In addition, the sample may be applied to the device and thenseparated into different reaction chambers, each with separatediagnostic elements. The capture zone can be various geometrical symbolsor letters to denote a code when the sample is positive or negative forthe target ligand. One skilled in the art will recognize the usefulcombinations of the elements of this invention.

The diagnostic element can also be configured to perform asemi-quantitative or quantitative assay, as for example, is described inClinical Chemistry (1993) 39, 619-624, herein referred to by referenceonly. This format utilizes a competitive binding of antigen and antigenlabel along a solid phase membrane. The improvement is that the use ofthe diagnostic element described herein for the above cited method wouldrequire a smaller sample volume and improved binding efficiency to thesolid phase surface.

Diagnostic Elements Other Than Capillaries

The inventive teachings described herein of the adsorption of proteins,particularly receptors to plastic surfaces, can be utilized foradsorption of receptors to many plastic surfaces which are not a part ofa capillary. Nanoparticles and latex particles coated with receptors canalso be applied to surfaces of many types of immunoassay devices, suchas, to “dipsticks” or lidless devices. For example, dipsticks aregenerally a solid phase onto which are bound, as a result of the assayprocess, for example, the ligand receptor conjugate. Dipsticks generallyincorporate membranes; however, a disadvantage in the use of membranesin dipsticks is the difficulty in washing the unbound ligand receptorfrom the membrane. Thus, an improvement in the use of dipsticks is toimmobilize receptor coated latex or nanoparticles directly onto aplastic surface of the dipstick. The removal of unbound ligand conjugatefrom the plastic surface is thus more efficient than removal from amembrane.

Textured surfaces such as disclosed herein can be used in diagnosticelements other than capillaries. In such embodiments, a textured surfacecan serve to provide additional surface area which allows for a higherdensity of assay reagents to be immobilized thereon. Furthermore, atextured surface, or other surface modifications, can be provided toaffect the flow characteristics of a fluid on or within the surface. Forexample, as disclosed herein a surface can be provided with hydrophobicregions to diminish the extent of fluid flow in the hydrophobic region,textures can be used that provide for a more uniform distribution ofdried reagents on the surface, textures can be provided to modify theconfiguration of the meniscus at the fluid flow front, or textures canbe used that provide the capillary driving force for movement of fluidwithin the surface.

Used Reagent Reservoir

Referring to FIGS. 1 and 2, the used reagent reservoir 7 receives thereaction mixture, other reagents and excess sample from the diagnosticelement 6. The volume of the used reagent reservoir 7 is at least thevolume of the sample and extra reagents which are added to or are in thedevice. The used reagent reservoir 7 can take many forms using anabsorbent, such as a bibulous material of nitrocellulose, porouspolyethylene or polypropylene and the like or the used reagent reservoircan be comprised of a series of capillary grooves. In the case ofgrooves in the used reagent reservoir 7, the capillary grooves can bedesigned to have different capillary pressures to pull the reagentsthrough the device or to allow the reagents to be received without acapillary pull and prevent the reagents from flowing backwards throughthe device. The size and quantity of the grooved capillaries determinethe volume and capillarity of the used reagent reservoir 7. In apreferred embodiment, as shown in FIG. 4, the fingers 52 at the end ofthe diagnostic element 6 are in fluid contact with a capillary space 55and the capillary space 55 is in fluid contact with a grooved ortextured capillary space 56. The depth of the grooves or texturedsurface can be, for example, about 0.1 mm to 0.6 mm, preferably about0.3 mm to 0.5 mm and the density can range from about 5 to 75 groovesper cm and preferably about 10 to 50 grooves per cm.

Referring to FIG. 4, the reagents of the device move to the fingers 52at the end of the diagnostic element 51 and into the capillary channel55. The reagents either partially or completely fill the capillary space55 and then come in contact with the grooved or textured surface 56. Thewidth of the capillary space 55 is generally about 1 mm to 3 mm and thedepth is generally about 0.1 mm to 2 mm. The length of the capillaryspace 55 should be sufficient to be in fluid contact with the grooved ortextured surface 56. The grooved or textured surface 56 partially orcompletely pulls the reagents from the capillary channel 55 depending onthe rate of delivery of the reagents into the capillary space 55 fromthe diagnostic element 51. When the flow of reagents is complete in thedevice, the grooved or textured surface 56 has greater capillarity thanthe capillary channel 55 and the reagents are removed from the capillarychannel 55 by the grooved or textured surface 56. In addition, thereverse flow of the reagents from the grooved or textured surface is notpreferred because the capillarity in the grooved or textured surface 56holds the reagents and prevents their backward flow. One skilled in theart can recognize from these inventive features that the arrangement ofgrooves or a used reagent reservoir within the device can be adapted toa variety of desired objectives.

The Description of the One-Step Assay Device

The elements of the device which have been described individually can beassembled in various ways to achieve the desired function. The term“one-step” implies that one manual action is required to achieve theassay result, for example, adding sample to the device is one step. Inthe case of the device performing a one-step assay which involves both atimed incubation of reagents and a wash step, the wash solution isexcess sample and the assay device is built with the elements in fluidcommunication using the sample addition reservoir, the sample-reactionbarrier, the reaction chamber, the time gate, the diagnostic element andthe used reagent reservoir as depicted in FIG. 1. The devices aregenerally about 3 cm to 10 cm in length, 1 cm to 4 cm in width and about2 mm to 15 mm thick. Typically, a top member with smooth surfaces isplaced onto a bottom member which has a surface onto which are built theelements stated above. The relationship of the elements are as depictedin FIG. 1. The reagents required for performing the assay areimmobilized or placed in the respective elements. The surfaces arebrought together, a capillary distance apart, and in doing so, theregions of the sample addition reservoir, the sample reaction barrier,the reaction chamber, the time gate, the diagnostic element, the gap andthe used reagent reservoir are all formed and are capable of functioningtogether. Also, the surfaces are brought together such that the opposingsurfaces touch to form and seal the sample addition reservoir, thereaction chamber, and the used reagent reservoir.

When performing a qualitative, non-competitive assay on one or moretarget ligands, the signal producing reagents, which could include, forexample, a receptor specific for the target ligand adsorbed to acolloidal metal, such as a gold or selenium sol, are placed on thesample reaction barrier or in the reaction chamber in dried orlyophilized form. Another receptor for each target ligand is immobilizedonto the surface of the diagnostic element at the capture zone. The timegate is positioned generally on the diagnostic element between thereaction chamber and the capture zones by the placement of, for example,a surfactant-free polystyrene suspension onto the device in an amountwhich dictates the desired incubation time. The incubation time isusually the amount of time for the reactions to come to substantialequilibrium binding. The assay is then performed by addition of sampleto the sample addition reservoir of the device. The sample moves overthe sample-reaction barrier, into the reaction chamber by the aid of thefingers and dissolves the reagents in the reaction chamber to form thereaction mixture. The reaction mixture incubates for the amount of timedictated by the time gate. The excess sample remaining in the sampleaddition reservoir and reaction mixture in the reaction chamber are influid communication but are not in substantial chemical communicationbecause of the sample-reaction barrier. Thus, the reaction chamberdefines the volume of the reaction mixture. The reaction mixture thenmoves past the time gate and onto the diagnostic element and over thecapture zones. The complex of receptor conjugate and target ligandformed in the reaction mixture binds to the respective receptor at thecapture zone as the reaction mixture flows over the capture zones. Thereaction mixture may also flow over a positive control zone, which canbe for example, an immobilized receptor to the signal developmentelement. As the reaction mixture flows through the diagnostic elementand into the used reagent reservoir by the aid of the fingers, theexcess sample flows behind the reaction mixture and generally does notsubstantially mix with the reaction mixture. The excess sample movesonto the diagnostic element and removes the receptor conjugate which didnot bind to the capture zone. When sufficient excess sample washes thediagnostic element, the signal at the capture zones can be interpretedvisually or instrumentally. Referring to FIG. 1D, in a preferred mode ofthe above description, the reaction mixture moves onto the diagnosticelement 6, over the capture zone or zones and then the reaction mixtureproceeds into a capillary gap 18. The capillary gap 18 generally hasless capillarity than that of the diagnostic element 6. The capillaryspace 19 of the diagnostic element 6 is generally smaller than thecapillary space of the gap 18. The volume of the capillary gap 18generally approximates the volume of the reaction mixture such that thecapillary gap 18 fills slowly with the reaction mixture and once filled,the capillarity of the remaining portion of the diagnostic element 6 orused reagent reservoir is greater than the capillarity of the gap 18resulting in an increased rate of flow to wash the diagnostic element 6.As one skilled in the art can appreciate, the gap 18 can be formed inthe top member 8 or in the bottom member 9 or a combination of bothmembers 8 and 9.

In the case of the device performing a one-step assay which does notinvolve a timed incubation step but does involve a wash step in whichthe wash solution is excess sample, the assay device is built with theelements in fluid communication using the sample addition reservoir, thesample-reaction barrier, the reaction chamber, the diagnostic elementand the used reagent reservoir. The assay reagents are used as describedabove for the non-competitive qualitative assay. The assay devicewithout the time gate allows the reaction mixture to flow onto thediagnostic element without an extended incubation time. The capillaryflow of the reaction mixture and the excess sample are as describedabove.

The optional reagent chamber is incorporated into the device in the caseof the device performing a one-step assay with the introduction of anadditional assay reagent into or after the reaction mixture or theintroduction of a wash solution which flows behind the reaction mixturethrough the device. The optional reagent chamber may be in fluid contactwith any element of the device and is generally in fluid contact withthe reaction chamber. When in fluid contact with, for example, thereaction chamber, the optional reagent chamber and the reaction chambermay be separated by a time gate. Various reagents may be dried orlyophilized in the optional reagent chamber, such as detergents for awashing step or reagents which are sequentially provided to thediagnostic element after the reaction mixture.

In the case of performing one-step, non-competitive, quantitative assaysthe reagents as described above for the non-competitive, qualitativeassay may apply. The device is comprised of the elements, sampleaddition reservoir, sample-addition barrier, reaction chamber, timegate, diagnostic element and used reagent reservoir. In this case, thecapture zone of the diagnostic element is generally the entirediagnostic element. That is, the capture zone is a length of thediagnostic element onto which the receptor conjugate binds. The receptorconjugate binds along the length of the capture zone in proportion tothe amount of target ligand in the sample. Alternatively, one or morecapture zones 17 can be placed on the diagnostic lane (FIG. 3A-B), andsignals from the capture zone(s) can be read by an instrument such as aCCD camera, a fluorometer or a spectrophotometer.

The device of the present invention is preferred for this quantitativeassay because of the high efficiency of capture of the reagents, forexample, the binding of a complex of target ligand and receptorconjugate to an immobilized receptor to the target ligand on the capturezone, and because the movement of the reaction mixture over thediagnostic element proceeds with a sharp front. The receptors on thecapture zone sequentially become saturated with the complex of targetligand and receptor conjugate as the reaction mixture moves over thelength of the capture zone. The length of the diagnostic elementcontaining bound conjugate then determines the concentration of thetarget ligand. Those skilled in the art will recognize the format ofthis type of immunoassay as a quantitative immunochromatographic assayas discussed in U.S. Pat. Nos. 4,883,688 and 4,945,205, herebyincorporated by reference.

In the case of the device performing a one-step, quantitative orqualitative competitive assay which involves both a timed incubation ofreagents and a wash step and the wash solution is excess sample, theassay device is built with the elements in fluid communication using thesample addition reservoir, the sample-reaction barrier, the reactionchamber, the time gate, the diagnostic element and the used reagentreservoir. When performing a qualitative competitive assay on one ormore target ligands, the conjugate is composed of, for example, a ligandanalogue coupled to signal development element, such as a gold orselenium sol. The conjugate and receptor for each target ligand areplaced in the reaction chamber in dried or lyophilized form, forexample, in amounts which are taught by U.S. Pat. Nos. 5,028,535 and5,089,391, hereby incorporated by reference. Another receptor for eachtarget ligand is immobilized onto the surface of the diagnostic elementat the capture zone. The time gate is positioned generally on thediagnostic element between the reaction chamber and the capture zones asdescribed previously. The incubation time is usually the amount of timefor the reactions to come to substantial equilibrium binding.

The assay is then performed by addition of sample to the device. Thesample moves over the sample-reaction barrier and into the reactionchamber, dissolves the reagents to form the reaction mixture andincubates for the time dictated by the time gate. The excess sample andreaction mixture are in fluid communication but not in substantialchemical communication because of the sample-reaction barrier. Thereaction mixture then moves onto the diagnostic element and over thecapture zones. The ligand analogue conjugate binds to the respectivereceptor or receptors at the capture zone or zones. As the reactionmixture flows over the diagnostic element and into the used reagentreservoir, the excess sample flows behind the reaction mixture andgenerally does not substantially mix with the reaction mixture. Theexcess sample moves onto the diagnostic element and removes conjugateswhich do not bind to the capture zone or zones. When sufficient excesssample washes the diagnostic element the results at the capture zonescan be interpreted visually or instrumentally.

In a preferred mode of the present invention, the reaction mixture movesonto the diagnostic element, over the capture zone or zones and then thereaction mixture proceeds into a capillary gap. The capillary gap hasless capillarity than that of the diagnostic element. The volume of thecapillary gap generally approximates the volume of the reaction mixturesuch that the capillary gap fills slowly with the reaction mixture andonce filled, the capillarity of the remaining portion of the diagnosticelement or used reagent reservoir is greater resulting in an increasedrate of flow of excess sample to wash the diagnostic element.

In another aspect of the one-step, competitive assay, the reactionmixture is composed of ligand analogue-ligand complement conjugate toeach target ligand and receptors adsorbed to latex particles withdiameters of, for example, 0.1 μm to 5 μm to each target ligand, inappropriate amounts, for example, as taught by U.S. Pat. Nos. 5,028,535and 5,089,391. The ligand complement on the conjugate can be anychemical or biochemical which does not bind to the receptors for thetarget ligands. The assay is begun by addition of sample to the device.Sample fills the reaction chamber and is incubated for a time whichallows the reagents to come to substantial equilibrium binding. Thereaction mixture flows over the time gate and onto or into a filterelement to prevent ligand analogue-ligand complement conjugates whichhave bound to their respective receptor latexes from passing onto thediagnostic element. Typical filter elements can be composed ofnitrocellulose, cellulose, nylon, and porous polypropylene andpolyethylene and the like. Thus, only the ligand analogue-ligandcomplements conjugate which were not bound by the receptor latex willpass onto the diagnostic element. The receptor to the ligand complementof the conjugate is immobilized on the diagnostic element at the capturezone and binds the conjugate. A wash step may not be required becausethe filter removes the conjugate bound to latex; however, the excesssample or a wash solution from the optional reagent chamber may be usedto wash the diagnostic element.

In the case of a one-step quantitative, competitive assay, the receptorto the ligand analogue conjugate or the ligand complement of theconjugate is immobilized onto the diagnostic element as describedpreviously for the one-step quantitative, non-competitive assay. Thus,the concentration of the target ligand in the sample is visualized bythe distance of migration on the diagnostic element of the conjugate. Inanother mode, a quantitative assay could be performed by the binding ofthe labeled conjugate, for example, the ligand analogue-ligandcomplement conjugate, to sequential, discrete capture zones of receptoron the diagnostic element. The quantitative result is achieved by thedepletion of the conjugate as the reaction mixture flows through thecapture zones of the diagnostic element. Signal related to analyteconcentration is measured, e.g., by a CCD camera, a fluorometer or aspectrophotometer.

The Device as a Diagnostic Element

The diagnostic element of the device can be utilized with a sampleaddition means to perform a separation step of bound and unboundconjugates. An example of this type of device which has a sampleaddition means, a diagnostic element and a used reagent reservoir isdepicted in FIG. 2. For example, in the case of a non-competitive assay,at least one receptor conjugate is incubated with sample which issuspected of containing at least one target ligand in a suitable vesseland this reaction mixture is applied to the sample addition zone of thedevice. The reaction mixture then flows onto the diagnostic element andover the capture zone of, for example, immobilized receptor to thetarget ligand. When target ligand is present in the sample, the targetligand-receptor conjugate complex binds to the receptor on the capturezone. If the signal development element is an enzyme, then either asubstrate for the enzyme which produces a visual color or a washsolution followed by a substrate is next added to the device. Excessreagents flow to the used reagent reservoir. The presence or amount ofeach target ligand in the sample is then determined either visually orinstrumentally.

In the case of a competitive immunoassay, for example as taught by U.S.Pat. Nos. 5,028,535 and 5,089,391, herein incorporated by reference, thediagnostic element may be used to separate bound and unbound ligandanalogue conjugates such that the unbound ligand analogue conjugatesbind to the receptors of the diagnostic element in proportion to thepresence or amount of target ligand in the sample.

One skilled in the art can appreciate that all formats of immunoassaysor nucleic acid assays which require a separation step of free and boundconjugates or the separation of free of bound reagents whichsubsequently leads to the ability to detect a signal can utilize theinventive features of the diagnostic element. One skilled in the art canalso recognize that the inventive elements of this invention, namely,the fingers, the sample reaction barrier, the reaction chamber, the timegate, the diagnostic element, the fluid control means and the usedreagent reservoir can be used separately or in various combinations andin conjunction with other devices not described here. Furthermore,textured surfaces, such as described herein, can be utilized in one ormore regions of the device to facilitate placement of a uniform layer ofdried reagent in the area, or to modify fluid flow characteristicsthrough the region. In addition, hydrophobic zones can be placed in aregion of the device to modify fluid flow characteristics in the region.As appreciated by one of ordinary skill in the art, features disclosedherein can be utilized in various combinations in the preparation anduse of assay devices.

For example, the sample reaction barrier with fingers and the reactionchamber can be used in conjunction with devices incorporating porousmembers, such as membranes to deliver precise volumes of reagents to theporous member. The time gate can also be incorporated into theaforementioned devices or the time gate may be used alone in conjunctionwith devices incorporating porous members. The fluid control means canalso be used in devices incorporating porous members to control the rateof flow of reagents through the porous member. In the context ofperformance of assays in accordance with the invention, channels canexist such as the distance between opposing walls of a particularregion, e.g., between the lid and the base; or the distance betweenadjacent texture structures. Accordingly, when a ligand receptor isimmobilized on a device surface, a ligand of interest in a sample candiffuse across the width of a channel to bind with its receptor.

EXAMPLES Example 1 Preparation of Anti-βhCG Antibody-Colloidal GoldConjugate

Colloidal gold with an average diameter of 45 nm was prepared accordingto the method of Frens, Nature, Physical Sciences, 241, 20 (1973). Thecolloidal gold conjugate was prepared by first adding 5.6 ml of 0.1 Mpotassium phosphate, pH 7.58, dropwise with rapid stirring to 50 ml ofcolloidal gold. Anti β-subunit monoclonal antibody to hCG (AppliedBiotech, San Diego, Calif.; 1 ml of 4.79 mg/ml in phosphate bufferedsaline, 0.02% sodium azide, pH 7) was added in a bolus to the colloidalgold with rapid stirring. After complete mixing the stirring was stoppedand the solution was incubated at room temperature for 1 h. Polyethyleneglycol (average molecular weight=20,000) was added (0.58 ml) as a 1%solution to the colloidal gold solution and the solution was mixed. Thecolloidal gold solution was subjected to centrifugation at 27,000 g and5 C. for 20 min. The supernatant was removed and each pellet was washedtwice by resuspension and centrifugation with 35 ml of 10 mM potassiumphosphate, 2 mM potassium borate, 0.01% polyethylene glycol (averagemolecular weight=20,000), pH 7. After the final centrifugation, thepellet was resuspended in 0.5 ml of the wash buffer. The gold conjugatewas diluted for the assay of hCG into a buffered solution containing 10mg/ml bovine serum albumin at pH 8.

Example 2 Preparation of anti- hCG Antibody Latex

Surfactant-free polystyrene particles (Interfacial Dynamics Corp.,Portland, Oreg.; 0.106 ml of 9.4% solids, 0.4 μm) was added whilevortexing to anti α-subunit hCG monoclonal antibody (Applied Biotech,San Diego, Calif.; 0.89 ml of 6.3 mg/ml in 0.1 M 2-(N-morpholino) ethanesulfonic acid, (MES), pH 5.5) and the suspension was incubated at roomtemperature for 15 min. The suspension was subjected to centrifugationto pellet the latex particles. The pellet was washed three times bycentrifugation and resuspension of the pellet with 10 mM MES, 0.1 mg/mltrehalose, pH 5.5. The final pellet was resuspended in the wash bufferat a solids concentration of 1%.

Example 3 Preparation of Goat Anti-Mouse Latex

Surfactant-free polystyrene particles (Interfacial Dynamics Corp.,Portland, Oreg.; 0.11 ml of 9.4% solids, 0.6 μm) were added whilevortexing to goat IgG antibody against mouse IgG (Jackson ImmunoResearchLaboratories, Inc.; 0.89 ml of 0.34 mg/ml in 0.1 M MES, pH 5) and thesuspension was incubated at 45° C. for 2 h. The suspension was subjectedto centrifugation to pellet the latex particles. The pellet was washedthree times by centrifugation and resuspension of the pellet with 10 mMMES, 0.2 mg/ml trehalose, pH 5.5. The final pellet was resuspended inthe wash buffer at a solids concentration of 1%.

Example 4 Preparation of the One-Step Device for a Qualitative orQuantitative hCG Assay

A one-step device made of plastic was built having an 80 to 100 μlsample addition reservoir, a 20 μl reaction chamber and a 40 μl usedreagent reservoir. This device is designed for applying samples of about20 μl to 100 μl, but the reaction chamber is fixed at 20 μl. In caseswhere a larger reaction mixture volume is required for the desiredassay, then the reaction chamber would be increased to that volume andthe sample addition reservoir would be about 2 to 4 times the volume ofthe reaction chamber volume.

The devices were plasma treated to graft functional groups which createa hydrophilic surface. Those skilled in the art will recognize that theplasma treatment of plastic is performed in a controlled atmosphere of aspecific gas in a high frequency field. The gas ionizes, generating freeradicals which react with the surface.

The sample addition reservoir was shaped as a trapezoid with dimensionsof 14 mm and 7 mm for the parallel sides and 7 mm for the other sideswith a depth of 0.49 mm. The sample addition reservoir was adjacent tothe sample reaction barrier.

The sample-reaction barrier was 1.5 mm long and 7 mm wide includinggrooves running parallel to the flow of the sample at a density of 50grooves per cm and a depth of 0.1 mm. In the case of sample volumeslarger than 20 to 80 μl, the width of the reaction barrier and therebythe reaction chamber could be increased to accommodate the desired flowrate but the groove size or density could remain as indicated.

The fingers in the walls of the reaction chamber and the used reagentreservoir were 1 mm wide and 0.4 mm deep with 7 fingers in each wall ofthe reaction chamber and the used reagent reservoir. The reactionchamber volume was 20 μl. The reaction chamber was shaped as a trapezoidwith dimensions of 7 mm and 3.5 mm for the parallel sides and 7.1 mm forthe other sides with depths of 0.56 mm for 20 μl reaction chambers.

The diagnostic element was about 2.5 cm long, 2 mm wide and 1 mm fromthe base of the device including grooves running perpendicular to theflow of reaction mixture at a density of 100 grooves per cm and a depthof 0.05 mm. In the case of a time gate on the diagnostic element, thetime gate was positioned on the diagnostic element immediately adjacentto the reaction chamber. The width of the diagnostic element could beincreased to increase the flow of the reaction mixture to the desiredrate past the capture zones.

The anti-αhCG antibody latex (1 μl) and the goat anti-mouse latex (1 μl)were applied to the diagnostic element of the devices approximately 1.5cm apart. The anti-βhCG antibody colloidal gold conjugate (10 μl) waspipetted into the trough of the reaction chamber.

The devices were placed under vacuum for about 15 min. to dry thereagents. The used reagent reservoir had the shape of a trapezoid withdimensions of 7 mm and 15 mm for the parallel sides and 8 mm for theother sides with a depth of 0.5 mm.

Referring to FIG. 4, in a preferred embodiment of the used reagentreservoir, the reaction mixture moved to a capillary space 55 (1.25 mmlong, 27.5 mm wide and 0.48 mm deep) from the diagnostic element 6,aided by fingers 52 (1 mm wide and 0.4 mm deep with 7 fingers), and theninto a grooved capillary structure (13.6 mm long, 25.4 mm wide, 0.61 mmdeep with a density of 16 grooves per cm). The outer walls and the topsurface of the walls of the sample addition reservoir and the reactionchamber had applied a thin coating of silicon grease to prevent theleakage of the reagents from the reservoir and chamber of the assembleddevice. The capillary spaces in the devices were then formed by placinga clear plastic polycarbonate sheet on top of the device. The plasticsheet was held to the opposing surface with binder clips. The clearplastic sheet had a sample port above the sample addition reservoir forthe introduction of sample.

Example 5 Hydrophobic Borders to Fluid-Containing Areas

Fluid flow on a surface or in a capillary is affected by the surfacetension of the fluid. For example, in a capillary channel that is formedby essentially planar walls that intersect along corners, fluid flowpreferentially precedes along the corners. The predisposition for fluidflow to proceed at corners occurs because the corners of a capillarycreate the lowest surface tension for the fluid.

However, when a uniform flow front is required within a capillary, thereduced surface tension at corners of the capillary can cause anon-uniform flow front. Non-uniform flow fronts can result in thecreation of air pockets within the capillary. If air pockets occur,wetting of the capillary surfaces within the air pocket is impaired orprevented. Consequently, when surfaces of capillaries are used, forreactions such as binding of antibodies or antigens, for chemicalreactions, or for nucleic acid hybridization reactions, the creation ofair pockets decreases the efficiency of the reaction. Furthermore, thecreation of air pockets within analogous capillaries of individualdevices of the same design is not predictable, consequently theconsistency of binding or chemical reactions between the individualdevices will be poor. Thus, air pockets within capillaries can alterfluid flow or even prevent it in the capillary.

Embodiments of this invention which comprise hydrophobic areas on alumenal surface of a capillary space act to control fluid flow withincapillaries, and more specifically to minimize fluid flow at the cornersof capillaries so that the fluid flow front is convex rather thanconcave.

The inventive teachings herein show that hydrophobic borders liningcapillary channels, preferably along edges or at corners, slows fluidflow at these locations, thereby creating convex flow fronts instead ofthe native concave flow front. Concave flow fronts are disadvantageousin capillary channels because air can be trapped as the concave flowfront proceeds through the capillary, since a concave flow frontincreases the propensity of the advancing fluid to form air pockets inthe capillary. Hydrophobic borders facilitate the escape of air from theadvancing fluid flow front because the likelihood is substantiallyreduced that fluid can be held within the capillary in the hydrophobiczones.

In a preferred embodiment, hydrophobic zones are applied to a surface.Specifically, a hydrophobic zone can be located on at least one surfaceof a capillary, each hydrophobic zone bordering the edge or comer of thecapillary, being located adjacent a hydrophilic surface in which fluidis intended to flow. On at least one surface of the capillary, thehydrophobic zones or borders occupy between 1% to 90% of the surface,each zone being adjacent to a hydrophilic surface and the edge or cornerof the capillary.

Hydrophobic zones delimit the edges of a surface or occupy the edges ofmaterials placed in capillary spaces. In another preferred embodiment,hydrophobic zones delimit the edges of materials placed in capillaryspaces, for example, materials such as filters, membranes and polymericmeshes. The hydrophobic zone can cover from 1% to 90% of the surface ofthe material to be placed in the capillary space. For additionaldisclosure concerning the use of materials within capillary spaces, seee.g., copending U.S. application Ser. No. 08/704,804 filed Aug. 26,1996, which is incorporated by reference herein. Accordingly, fluid flowin the capillary is delayed at the edges of the material in contact withthe corners of the capillary relative to the fluid flow within thehydrophilic zones of the capillary. The hydrophobic zones of thematerial in the capillary occupy between 1% and 90% of the surface ofthe material, each zone being adjacent to the hydrophilic surface andthe edge of the capillary.

In addition, embodiments of the invention allow for the application offluids to discrete zones on surfaces or within surfaces or membranes.Thus, in another preferred embodiment hydrophobic zones of surfacesprevent the movement of reagents on or within a surface or within amembrane. In this embodiment, these zones act as corrals to hold fluidwithin an area of the surface. These embodiments overcome a difficultyof applying reagents to discrete zones on or within surfaces or inmembranes, such that a volume of applied reagent will move on a surfaceor within a membrane because of the hydrostatic pressure of the reagent.This problem is especially prevalent in the case of surfaces whichcomprise a texture to facilitate movement of fluids by capillary actionalong the surface such as during the performance of an assay on a fluidsample. However, when manufacturing such assay devices, it can bedesirable to place reagents on such surfaces, application of discretezones of reagents is especially difficult because the surface isdesigned to facilitate fluid movement by capillary action, and theeffects of hydrostatic forces exacerbate the difficulties produced bycapillary action. These factors create an unpredictable area of reagent,which may not be discrete. Consequently, if the volume of the appliedreagent relative to the surface is too great, adjacent reagent areas mayrun together to create one undesired, commingled zone. This situation isparticularly problematic when the surface comprises grooves or texturethat cause fluids to flow by capillarity on or within the surface.Generally, such surfaces are substantially fluid impermeable.Accordingly, the creation of hydrophobic borders on or within a surfaceor in a membrane to encompass or retain the applied reagent allows theapplication of reagents in discrete areas.

In another preferred embodiment, hydrophobic zones are applied tosurfaces to control the overall movement of the fluid on or within asurface or in a capillary. For example, hydrophobic zones can beutilized to direct or prevent fluid flow to various areas of a device.

In an alternative preferred embodiment, hydrophobic borders are placedat the edge(s) of a surface such as adjacent to corners of a chamber, tofacilitate uniform drying of liquids on the surface. This embodiment isuseful in the drying of liquid on a surface, wherein, in the absence ofhydrophobic borders, liquid which is added to the surface or chamberaccumulates at the edge or corners of the surface or chamber asevaporation occurs. This latter situation occurs because the corners ofa chamber create a meniscus and it is energetically favorable for afluid to move to a corner meniscus as it evaporates. Therefore, theoutcome of evaporation is a disproportionately larger amount of driedreagent in the corners of the chamber than in the surface of thechamber. The novel use of hydrophobic borders adjacent to corners ofchambers prevents the fluid meniscus from forming at the comer, sincethe fluid will not accumulate at the hydrophobic border. Thus, by use ofthis embodiment, the resultant dried liquid is more uniformly dispersedon the floor of the chamber.

Hydrophobic zones can also be created for surfaces which had previouslybeen made hydrophilic. Several techniques are known to those skilled inthe art for use to make a surface hydrophilic, for example, coronadischarge, treating with a gas plasma, treating with detergents orproteins and the like. For surfaces made hydrophilic by theaforementioned techniques, hydrophobic zones can be created byapplication of organic solvents that destroy the plasma treatment ordenature the proteins, to recreate a native hydrophobic plastic surfaceor to create a hydrophobic surface by the denatured proteins, or bylocal heating of the surface using focused laser beams to destroy thehydrophilic nature of the surface. Alternatively, one can maskhydrophobic areas before creating a hydrophilic area by any of theforegoing methods. The areas can be masked by objects such as a templateor can be masked by materials that are applied to the surface and thenare subsequently removed.

Hydrophobic compounds, such as aliphatic and/or aromatic compounds andvarious inks and polymers and the like can be used for the creation ofhydrophobic zones in accordance with the invention. The compounds aregenerally dissolved in organic solvents or mixtures of aqueous andorganic solvents. One skilled in the art will recognize that a varietyof techniques known in the art (such as ink jet printing, spraying, silkscreening, drawing, embossing and the like) are techniques that permitthe application of hydrophobic zones on or within surfaces.

Example 6 Textured Surfaces to Facilitate Uniform Drying of Reagents

In an additional embodiment, texture structures such as posts arepositioned, generally in an ordered array on a surface. When fluid isplaced in contact with the structures, small menisci are formed at eachstructure. When the reagent fluid dries, these menisci thereby provide avery uniform distribution of dried reagent on the surface. Generally,the structures are posts which are substantially perpendicular to asurface such as a floor of a chamber. A rectilinear angle is definedbetween a surface and a wall of a post located thereon. The density andsize and shape of posts on the surface can vary, depending on the degreeof uniformity desired for the dried reagents. Post height can also vary,and generally the height of the posts should be about 1% to more than100% of the height of the fluid in the chamber; that is, the posts canprotrude from the fluid or the fluid can cover the posts after applyingthe fluid to the surface. In all cases, the posts will act as zones toform menisci as liquid evaporates from the surface or chamber.

Example 7 Qualitative or Quantitative One-Step Assay for hCG

The devices described in Example 4 were used for the qualitative orquantitative one-step assay for hCG. The assay times for the deviceswithout the time gates were about 5 to 10 min. A urine solution (60 μl)containing 0, 50, 200 and 500 mIU hCG/ml was added to the samplereservoir of the devices. The sample moved into the reaction chamber,dissolved the colloidal gold conjugate and the reaction mixture movedonto the diagnostic element over the anti-hCG latex and goat anti-mouseIgG latex capture zones. The reaction mixture moved into the usedreagent reservoir and the excess sample washed the diagnostic element.The color density of the capture zones for hCG was measuredinstrumentally using a MINOLTA CHROMA METER CR 241 at 540 nm. A redcolor was visible for samples containing hCG and not visible for thesample without hCG at the capture zones for hCG. The _E* values for the0, 50, 200 and 500 mIU/ml were 0, 7.78, 12.95 and 20.96, respectively,and for the positive control (goat anti-mouse IgG) zones a distinctivered bar was observed with a _E* of about 35.

Example 8 Qualitative or Quantitative One-Step Assay for hCG Using aTime Gate

Devices as described in Example 4 were prepared with the addition of thetime gate. The time gate was formed on the diagnostic element which isin contact with the reaction mixture in the reaction chamber.

The time gate was prepared by adding 1 μl of 2% solids ofsurfactant-free, sulfated latex, 1.0 μm, (Interfacial Dynamics Corp.,Portland, Oreg.). The other reagent latexes and gold conjugate were alsoadded to the devices and dried as described in Example 7. Clear plasticsheets were placed on the devices and various aliquots of sample (about60 μl) containing 0, 50, 200 and 500 mIU hCG/ml, respectively, was addedto the devices.

The sample moved into the reaction chamber, dissolved the colloidal goldconjugate and the reaction mixture remained in the reaction chamber forabout 8 to 10 min, whereas in devices without time gates the reactionmixture remained in the reaction chamber for 5 sec to 15 sec. Theproteinaceous components of the reaction mixture, which may be presentin the sample and which was added as a component of the reactionmixture, namely, bovine serum albumin, bound to the latex particles ofthe time gate and changed the hydrophobic surface of the time gate intoa hydrophilic surface. Other proteins, such as gelatin, serum albumins,immunoglobulins, enzymes and the like and polypeptides and hydrophilicpolymers will also function to bind to the hydrophobic zone.

The gradual transformation of the hydrophobic surface to a hydrophilicsurface, which resulted through binding of the proteinaceous componentsof the reaction mixture to the latex particles allowed the reactionmixture to flow over the area of the time gate.

In control experiments in which protein, namely bovine serum albumin,was not added to the reaction mixture, flow of the reaction mixture overthe time gate and onto the diagnostic element did not occur during thetime (5 h) of the experiment. This control experiment showed that theurine sample alone did not contain sufficient protein or componentswhich bind to the applied latex of the time gate to allow a change inthe hydrophobic character of the time gate. In the event that thecomponents in the sample should only be used to cause the transformationof the hydrophobic time gate to a hydrophilic one for the reactionmixture to flow, then one would be required to lower the mass and totalsurface area of the latex applied to the time gate to an extent whichwould allow flow of the reaction mixture over the time gate in anappropriate amount of time.

The reaction mixture then moved onto the diagnostic element over theanti-hCG latex and goat anti-mouse IgG latex capture zones. The reactionmixture moved into the used reagent reservoir and the excess samplewashed the diagnostic element. The color density of the capture zonesfor hCG was measured instrumentally using a MINOLTA CHROMA METER CR 241.A red color was visible for samples containing hCG and not visible forthe sample without hCG at the capture zones for hCG. The _E* values forthe 0, 50, 200 and 500 mIU/ml were 0, 6.51, 13.14 and 18.19,respectively. A red color bar was visible at the goat anti-mouse IgGcapture zones of each device.

Example 9 Qualitative or Quantitative One-Step Assay for hCG Using aFlow Control Means

Devices as described in Example 4 were prepared with the addition of theoptional flow control means.

The optional flow control means or “gap” was placed behind the capturezone for hCG gold conjugate on the diagnostic element. The gap betweenthe two surfaces was 0.38 mm, the length of the gap was 13.2 mm and thewidth of the gap on the top member was 9 mm; however, the effectivewidth of the gap was the width of the diagnostic element (2 mm). Thisgap volume above the diagnostic element was about 10 μl which was, inthis case, half the volume of the reaction chamber.

The anti-hCG and the goat anti-mouse latexes and gold conjugate wereadded to the device and dried as described in Example 7. Clear plasticsheets of polycarbonate having a gap in one surface were placed on thedevices with the gap facing the diagnostic element. Sample (about 60 μl)containing 0 and 200 mIU hCG/ml was added to the devices. The samplemoved into the reaction chamber, dissolved the colloidal gold conjugateand the reaction mixture then moved onto the diagnostic element over theanti-hCG latex. The reaction mixture then entered the gap which wasimmediately behind the capture zone of anti-hCG latex. The flow rateover the capture zone slowed while the reaction mixture moved over thecapture zone and filled the gap. The time for the 10 μl reaction mixtureto fill the gap was about 12 min to 16 min, whereas with devices withoutthe optional flow control means, the times were about 1 min to 3 min.for the reaction mixture to pass over the capture zone. When thereaction mixture filled the gap, the reaction mixture then moved intothe narrow capillary of the diagnostic element and over the goatanti-mouse capture zone. The reaction mixture moved into the usedreagent reservoir and the excess sample washed the diagnostic element.

The color density of the capture zones for hCG was measuredinstrumentally using a MINOLTA CHROMA METER CR 241. A red color wasvisible for samples containing hCG and not visible for the samplewithout hCG at the capture zones for hCG. The _E* values for the 0 and200 mIU/ml were 0 and 16.12. The E* value of the hCG capture zone forthe device without the flow control means for the 200 mIU/ml sample was16.32. A red color bar was visible at the goat anti-mouse IgG capturezones of each device.

Example 10 Preparation of the Diagnostic Element for Multi-step Assays

A device was built comprising a sample addition reservoir and adiagnostic element. The devices were plasma treated to graft functionalgroups which create a hydrophilic surface. The sample addition reservoirhad dimensions of 12 mm long, 6 mm wide and 0.05 mm deep. The diagnosticelement was about 5.5 cm long, 1.3 mm wide and 1 mm from the base of thedevice and included grooves running perpendicular to the flow ofreaction mixture at a density of 100 grooves per cm and a depth of 0.05mm. In the case of qualitative assays, the antibody latex (1 μl) wasapplied to the diagnostic element, covering the entire width and 1 cmlength of the diagnostic element. In the case of animmunochromatographic assay, the antibody latex (6 μl) was applied tothe entire width and length of the diagnostic element. The devices wereplaced under vacuum for about 1 h to dry the reagents. The capillaryspaces in the device were then formed by placing a clear plasticpolystyrene sheet on top of the device. The plastic sheet was held tothe opposing surface with binder clips.

Example 11 Assay for hCG Using the Diagnostic Element

The diagnostic element described in Example 10 was used for the assay ofhCG. Urine samples (20 μl) containing 0, 50, 200 and 500 mIU/ml hCG wereadded to tubes containing anti-βhCG antibody colloidal gold conjugate (2μl). The tubes were vortexed and the reaction mixtures were incubatedfor 5 min at room temperature. The reaction mixtures (20 μl) wereapplied in 10 μl aliquots to the sample addition reservoir of thedevice. The reaction mixture flowed onto the diagnostic element from thesample reservoir and over the capture zone. An absorbent at the end ofthe capture zone removed the used reagent from the diagnostic element.The color density of the capture zones for hCG was measuredinstrumentally using a MINOLTA CHROMA METER CR 241. A red color wasvisible for samples containing hCG and not visible for the samplewithout hCG at the capture zones for hCG. The _E* values for the 0, 50,250 and 500 mIU/ml were 0.00, 1.24, 3.16 and 5.56, respectively.

Example 12 Synthesis of meta-Nitrophencyclidine

To an ice cooled solution of phencyclidine hydrochloride (5 g, 1.8×10⁻²mol) in concentrated sulfuric acid (9 ml) was added dropwise, and withstirring, fuming nitric acid (2 ml). The reaction mixture was stirred inan ice-water bath for 1 hour and then poured onto crushed ice/water. Themixture was made basic with 10N sodium hydroxide (50 ml) to pH 12 andextracted with diethyl ether (2×100 ml). The combined organic layerswere washed with water (2×100 ml), dried over anhydrous magnesiumsulfate, filtered and evaporated under vacuum. The residue was treatedwith methyl alcohol (20 ml) and heated on a hot water bath (80° C.)until solute dissolved. The flask was covered with aluminum foil(product is light sensitive) and the solution was allowed to stir atroom temperature overnight when a yellow solid precipitated. The solidwas collected by filtration and dried under vacuum to afford 3.0 g (58%)of m-nitrophencyclidine as fine yellow crystals which were protectedfrom light: mp 81-82° C.

Example 13 Synthesis of meta-Aminophencyclidine

To a stirring solution of m-nitrophencyclidine (3.0 g, 10.4×10⁻³ mol) inmethyl alcohol (150 ml) was added, under a flow of argon, 10%palladium-carbon (0.5 g) followed by ammonium formate (4.0 g, 6.3×10⁻²mol). The reaction mixture was stirred at room temperature for 2 hoursafter which time the catalyst was removed by filtration and the solventwas evaporated under vacuum. The residue was treated with 1 N potassiumhydroxide solution (30 ml) and extracted with diethyl ether (2×50 ml).The combined organic extracts were washed with water (50 ml), dried overanhydrous magnesium sulfate, filtered and evaporated under vacuum. Theresidue was dissolved in hexane (20 ml) and the solution was stirred atroom temperature overnight when a white solid precipitated. The solidwas collected by filtration and dried under vacuum to afford 1.4 g (52%)of m-aminophencyclidine: mp 121-122° C.

Example 14 Synthesis of Acetylthiopropionic Acid

To a stirred solution of 3-mercaptoproprionic acid (7 ml, 0.08 moles)and imidazole (5.4 g, 0.08 moles) in tetrahydrofuran (THF, 700 ml) wasadded dropwise over 15 minutes, under argon, a solution of 1-acetylimidazole (9.6 g, 0.087 moles) in THF (100 ml). The solution was allowedto stir a further 3 hours at room temperature after which time the TEFwas removed in vacuo. The residue was treated with ice-cold water (18ml) and the resulting solution acidified with ice-cold concentrated HCl(14.5 ml) to pH 1.5-2. The mixture was extracted with water (2×50 ml),dried over magnesium sulfate and evaporated. The residual crude yellowoily solid product (10.5 g) was recrystallized from chloroform-hexane toafford 4.8 g (41% yield) acetylthiopropionic acid as a white solid witha melting point of 44-45° C.

Example 15 Synthesis of meta-Acetylthiopropionamide Phencyclidine

To a stirring solution of m-aminophencyclidine (1.4 g, 5.4×10⁻³ mol) andacetylthiopropionic acid (0.87 g, 5.8×10⁻³ mol) in anhydroustetrahydrofuran (7 ml) was added dicyclohexylcarbodiimide (1.19 g,5.8×10⁻³ mol). The flask was purged with argon and the solution stirredat room temperature for 2 hours. The mixture was filtered from insolubledicyclohexylurea and evaporated under vacuum. The residual solid wasrecrystallized from chloroform/hexane to afford 1.5 g (71%) ofm-acetylthiopropionamide phencyclidine as a white crystalline solid: mp152-4 C.

Example 16 Synthesis of meta-3-Mercaptoproprionamide Phencyclidine

meta-Acetylthiopropionamide phencyclidine (0.01 g, 2.57×10⁻⁵ mol) wasdissolved in 1.29 ml 0.12 M potassium carbonate in 80% methanol/20%water (v/v). The solution sat at room temperature for 5 min and then 0.2ml 0.5 M potassium phosphate, pH 7, was immediately added and thesolution was adjusted to pH 7-7.5 with hydrochloric acid (1 N). Thetitle compound in solution was used as is to react with BSA-SMCC.

Example 17 Preparation of Phencyclidine Analogue Attached to BovineSerum Albumin (BSA-PCP)

Bovine serum albumin (BSA, 3.5 ml of 20 mg/ml) was reacted withsuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC,Pierce Chemical Co.) by adding a solution of 6.7 mg SMCC in 0.3 mlacetonitrile and stirring the solution at room temperature for 1 h whilemaintaining the pH between 7 and 7.5 with 1 N potassium hydroxide. Theprotein was separated from unreacted compounds by gel filtrationchromatography in 0.1 M potassium phosphate, 0.02 M potassium borate,0.15 M sodium chloride, pH 7.0. The meta-3-mercaptoproprionamidephencyclidine (0.2 ml of 13 mM) was added to the BSA-maleimide (2 ml at8.2 mg/ml) and the solution was stirred at room temperature for 4 h. Thesolution was then dialyzed 3 times against 1000 ml of 10 mM MES, pH 5.5.Recover 1.8 ml BSA-PCP at 8 mg/ml.

Example 18 Preparation of Phencyclidine Analogue Colloidal GoldConjugate

A solution (4.7 ml) containing BSA (22 mg) and BSA-PCP (5.6 mg) in 10 mMMES, pH 5.5 was added in a bolus to colloidal gold (105 ml) in 10 mMMES, pH 5.5 with rapid stirring. After complete mixing the stirring wasstopped and the solution was incubated at room temperature for 1 h. Thecolloidal gold conjugate was subjected to diafiltration against 50 mMpotassium phosphate, 10 mM potassium borate, pH 7, using a tangentialflow device (Sartorius Easy Flow, molecular weight cutoff was 100,000)to remove BSA and BSA-PCP which was not bound to colloidal gold. Thegold conjugate was diluted for the assay of PCP into a buffered solutioncontaining 10 mg/ml bovine serum albumin at pH 7.5.

Example 19 Preparation of anti-Phencyclidine Antibody Latex

Surfactant-free polystyrene particles (Interfacial Dynamics Corp.,Portland, Oreg.; 0.074 ml of 9.4% solids, 0.4 μm) was added whilevortexing to anti-phencyclidine monoclonal antibody (0.926 ml of 5.86mg/ml in 0.1 M MES, pH 5) and the suspension was incubated at 45° C. for2 h. The suspension was subjected to centrifugation to pellet the latexparticles. The pellet was washed three times by centrifugation andresuspension of the pellet with 10 mM MES, 0.1 mg/ml trehalose, pH 5.5.The final pellet was resuspended in the wash buffer at a solidsconcentration of 1%.

Example 20 Preparation of Latex-Immobilized Affinity-Purified Goat IgGAntibody Against the Fc Fragment of Mouse IgG (Goat anti-mouse Fc latex)

Affinity-purified goat anti-mouse (Fc (Immunosearch) and polystyrenelatex particles (sulfated, 1.07 μm) (Interfacial Dynamics) wereincubated separately at 45° C. for one hour, the antibody solution beingbuffered with 0.1 M 2-(N-morpholino) ethane sulfonic acid at pH 5.5.While vortexing the antibody solution, the suspension of latex particleswas added to the antibody solution such that the final concentration ofantibody was 0.3 mg/ml and the solution contained 1% latex solids. Thesuspension was incubated for 2 hours at 45° C. prior to centrifugationof the suspension to pellet the latex particles. The latex pellet wasresuspended in 1% bovine serum albumin in phosphate-buffered-saline(PBS) and incubated for one hour at room temperature. Followingcentrifugation to pellet the latex, the pellet was washed three times byresuspension in PBS and centrifugation. The final pellet was resuspendedin PBS containing 0.1% sodium azide at pH 7.0 at a latex concentrationof 1% solids.

Example 21 Assay for Phencyclidine Using the Diagnostic Element

The diagnostic element described in Example 10 was used for the assay ofphencyclidine (PCP). Urine samples (133 μl) containing 0, 100, 200 and300 ng/ml PCP were added to tubes containing a lyophilized bufferformulation (containing 10 mM potassium phosphate, 150 mM sodiumchloride and 10 mg/ml BSA, pH 8) and phencyclidine analogue colloidalgold conjugate (4 μl) was added and the solution was vortexed. Anti-PCPantibody (2.8 μl of 0.1 mg/ml) was added to each tube and the solutionswere vortexed and incubated at room temperature for 5 min. Goatanti-mouse Fc latex (50 ml of a 1% suspension) was added to the tubes,the tubes were vortexed and incubated at room temperature for 10 min.The solutions were then filtered to remove the complex of the PCPanalogue gold conjugate:anti-PCP antibody:goat anti-mouse latex from thereaction mixture using a GELMAN ACRODISC® 3 syringe filter (0.45 μm).The filtrates of the reaction mixtures (20 μl) were applied to thediagnostic elements described in example 10. The reaction mixture flowedonto the diagnostic element from the sample reservoir and over thecapture zone. An absorbent tissue placed 1 cm after the capture zoneremoved the used reagent from the diagnostic element. The color densityof the capture zones was measured instrumentally using a MINOLTA CHROMAMETER CR 241. The _E* values for the 0, 100, 200 and 300 ng/ml sampleswere 0.69, 9.28, 14.04 and 21.6, respectively.

Example 22 Exemplary Device Configurations

A presently preferred mode of the invention utilizes a device embodimentcapable of performing one-step immunoassays. The device preferablycomprises a sample addition reservoir 1, a sample reaction barrier 3, areaction chamber 4, a time gate 5, a diagnostic lane 6, a used reagentreservoir 7, and a lid 64. FIG. 11 depicts a preferred embodiment of thedevice where the lid is removed to permit illustration of variousportions of the device. A lid is not illustrated in FIG. 11, asappreciated by one of ordinary skill in the art, the lid has an accessport so that fluid can be introduced into the sample addition reservoir;the lid can also have a vent to facilitate escape of gas as the devicefills with liquid. In one embodiment, a vent is located in the lid at anarea of the used reagent reservoir.

The sample addition reservoir can comprise a filter (not illustrated)for the separation of plasma from red blood cells or for the separationof debris in the sample from the sample to be assayed and a reservoirfor the storage of sample used in the assay device. For additionaldisclosure concerning filters, see e.g., copending U.S. application Ser.No. 08/704,804, filed Aug. 26, 1996 which is incorporated by referenceherein. The sample addition reservoir is in fluid communication with thesample reaction barrier.

The sample reaction barrier can comprise a texture composed of texturestructures on a surface thereof. Preferred texture height is about 0.01to 0.02 mm and width of each texture structure is about 0.09 to 0.20 mm.The distance between adjacent texture structures is about 0.080 to 0.100mm. The height of the capillary space in the sample reaction barrier isabout 0.02 to 0.08 mm. Preferably, the surface of the sample reactionbarrier at both edges of the capillary is made hydrophobic to preventfluid from preferentially flowing at the edges of the capillary. Sincethe hydrophobic surfaces minimize the flow along the edges of thereaction chamber, these surfaces also direct fluid flow into thereaction chamber, the access to which occurs toward the center of thesample reaction barrier. The sample reaction barrier preferablycomprises ten vertical grooves 16 that are in fluid communication withthe capillary spaces of the sample reaction barrier and the reactionchamber. The grooves are approximately 0.02 to 0.03 mm high and arespaced about 0.5 to 1.5 mm apart.

The reaction chamber is comprised of a capillary about 0.03 to 1.0 mmhigh and contains a volume of about 0.2 to 6 μl. Preferably, both innerlid and base surfaces of the reaction chamber capillary space comprise atexture of small texture structures, about 0.015 to 0.03 mm high, with adiameter of 0.05 to 0.1 mm, at a spacing of about 0.1 to 0.3 mm. Thereaction chamber is in fluid communication with the time gate. Onesurface of the reaction chamber adjacent to the time gate comprisesgrooves to define a flow front perpendicular to the direction of fluidflow. The grooves are oriented substantially perpendicular to thedirection of fluid flow. The grooves are generally 0.03 to 0.07 mm highand are spaced 0.08 to 0.12 mm apart. The surface at an edge of thereaction chamber, such as at a comer, is made hydrophobic. As disclosedherein, a hydrophobic region slows flow at the edges of the capillaryand prevents fluid from preferentially flowing at the edges.

The time gate is comprised of a capillary about 0.02 to 0.12 mm high.One surface of the time gate is comprised of grooves about 0.03 to 0.07mm high and spaced about 0.08 to 0.12 mm apart; these grooves arecontiguous with similar grooves in the reaction chamber. The grooves areoriented substantially perpendicular to the predominant direction offluid flow through the device. As disclosed herein, a surface of thetime gate is made hydrophobic to delay fluid flow out of the reactionchamber. The time gate is in fluid communication with the diagnosticlane.

The diagnostic lane/element comprises a capillary preferably about 0.01to 0.05 mm high and comprises a texture composed of texture structuresabout 0.01 to 0.02 mm high, 0.03 to 0.07 mm in diameter/width, andspaced about 0.04 to 0.09 mm apart. The volume of the diagnostic lane isabout 0.5 to 3 μl. The edges of the capillary in the diagnostic lane aremade hydrophobic to slow fluid flow at the edges of the capillary, andto prevent fluid from preferentially flowing at the edges of thecapillary. The diagnostic lane is in fluid communication with the usedreagent reservoir. As shown in FIG. 15, diagnostic lane 6 preferablybegins at a point 70. When the diagnostic lane begins at a point, thisallows fluid to enter the lane at a more predictable location, generallythe proximal-most location of the diagnostic lane. When fluid enters thelane in a predictable location, this in turn leads to increasedpredictability of fluid flow in the diagnostic lane itself which allowsfor uniformity of performance for devices of the same configuration.

The used reagent reservoir preferably comprises a capillary spacesimilar in dimension to the capillary of the diagnostic lane, and isgenerally of equal or greater volume. It is comprised of a texturecomprising texture structures that have a height of about 0.01 to 0.02mm, widths/diameters of about 0.03 to 0.07 mm which are spaced 0.04 to0.09 mm apart. The used reagent reservoir can comprise zones thatexhibit a color change upon addition of fluid, to visibly indicate tothe user that fluid has flowed past that particular zone. For example,the reservoir can contain colored zones that become colorless when fluidcomes into contact with them. These colored zones can be made of watersoluble dyes, such as green food coloring, that wash away throughdissolution by the advancing fluid. Alternatively, the region cancontain a zone that develops a color change when fluid has flowed in theregion. These zones can consist of receptors that bind a dye label inthe sample or bind an enzyme that generates color at the zone. Thesenovel color change features have application in indicating to the userof the test device the extent of completion of the procedure.

Referring now to FIG. 12, a device preferably comprises, generally atthe outer edge and in areas where capillary spaces of particulardimension are important, structures referred to as stops 60. The stopsserve to establish a capillary space of uniform height, e.g., amongvarious devices manufactured in the same way. A device also preferablycomprises energy directors 62. The energy directors also serve toestablish a capillary space of uniform height, e.g., among variousdevices manufactured in the same way, where an energy source such asultrasonic welding is used to join two parts by melting them together.The energy directors also function to join two pieces of a device, suchas joining a lid and a base. As depicted in FIG. 12, an energy directorhas a height greater than that of a stop. When stops and energydirectors are used together, the portion of the energy director that ishigher than a stop is induced to melt by an externally applied energysource. As such melting occurs, the two parts being joined come closertogether. The closeness of the approximation of the two parts is limitedby the stops which, when preferably are used with energy directors, donot melt and thus serve to define a uniform separation of two joinedparts. The uniform separation is a capillary space in preferredembodiments of a device in accordance with the invention.

Accordingly, the stops and energy directors are designed to define acapillary space and to maintain a stable union of a lid 64 to the base.Thus, adjacent to the sample addition reservoir, the sample reactionbarrier, the reaction chamber, the time gate and the diagnostic lane areenergy directors that adjoin the lid to the base, to complete theformation of capillary spaces within the device, and to seal fluid inthe capillary spaces. Typically, the lid is ultrasonically welded to thebase. Adjacent to the energy directors are stops that are about 0.02 to0.06 mm high. The stops act to prevent the lid from being attached tothe base in a manner that prevents formation of a capillary space; thestops thus serve to define a reproducible capillary space between manydevices. The stops are bordered by energy directors so that fluid doesnot enter the area of the stops.

Furthermore, as depicted in FIG. 11, the device preferably comprises oneor more regions of dead space 66 adjacent sides of the diagnostic lane.The dead space(s) allow for detection of a sensible signal, e.g., acolor change in the fluorescent or visible spectra, without interferencefrom any signal contained in reactants that are located in a usedreagent reservoir or other device region.

The novel use of stops, as described herein, serve to define capillariesor uniform height in devices. As appreciated by one of ordinary skill inthe art, the stop height can be varied to establish a variety ofcapillary spaces in a device. Furthermore, as illustrated in FIG. 12,various stop 60 and energy director 62 embodiments can be prepared inaccordance with the present invention. In general, the dimensions of acapillary space designed into a device is determined based on the natureof the sample to be assayed. For example, whole blood or lysed blood hasa higher viscosity than plasma or serum. Accordingly, devices weredesigned with higher capillary gaps to assay whole or lysed blood, thesedevices had higher gaps than devices for serum or plasma. When whole orlysed blood was used in assays in the devices with the higher capillarygaps, these devices achieved similar assay times and assaycharacteristics relative to devices configured for use with plasma orserum samples. The devices requiring higher capillary gaps have hadcorrespondingly higher stops.

The stops can be formulated using shims, layers of glue or hardeningagents, or they can be molded directly into the part, using injectionmolding or other conventional molding or fabrication processes. In thecase of using silicon chips, stops can be incorporated into devicesutilizing photolithography or micromachining techniques.

FIG. 13 depicts a electron micrograph of an embodiment of the inventionillustrating a sample addition reservoir 1, a textured sample reactionbarrier 3, a textured reaction chamber 4, a textured used reagentreservoir 7, a stop 60, and energy directors 62. In this embodimentenergy directors 62 and stop 60 collectively constitute a dead space.FIG. 14 is an enlarged view of a portion of FIG. 13, illustratingtextured sample reaction barrier 3, textured reaction chamber 4, anenergy director 62, and stop 60. FIG. 15 depicts an electron micrographof an embodiment of the invention illustrating a time gate 5, a textureddiagnostic lane 6, and an energy director 62. FIGS. 16 A-B depict twoviews of a textured surface in a capillary space adjacent an energydirector 62.

In a further preferred aspect of the invention, depicted in FIG. 11, thedevice is fabricated to have positioners 68, the lid (not shown) isfabricated to have positioning elements that mate with theasymmetrically placed positioners 68, to ensure that the lid is placedon the device with the correct orientation, so that the surface facingthe facing into the device has the appropriate texture. In addition, thelid has a hole at the region of sample addition chamber 1, to permitintroduction of fluid into the device.

In a preferred embodiment of an immunoassay device in accordance withthe invention, immunoassay reagents are placed on separate surfaceslocated in a given region of the device. For example, an immunoassayreagent can be immobilized on the lid in the area of the samplereservoir, sample reaction barrier, reaction chamber or diagnostic lane;and a separate immunoassay reagent can be immobilized on the base in anarea of the sample reservoir, sample reaction barrier, reaction chamberor diagnostic lane. It is particularly advantageous to place one reagenton a lid and another reagent on a base of the device when the lid andbase initially constituted separate pieces that are subsequentlyattached together in the manufacture of the device. One or more of suchimmobilized reagents can be diffusible when contacted by fluid.

In accordance with this embodiment of the invention, reagents that couldnot otherwise be packaged together in a capillary space of a devicewithout the occurrence of adverse cross reactions, can be placed in adevice in a single capillary space. For example, if a labeled antibodyand a capture antibody were placed together in a capillary space,non-specific interactions can occur in the absence of any targetmaterial. Such non-specific interactions lesson the sensitivity of anassay. Fundamental assay types that can utilize reagents localized onseparate surfaces in a capillary space include, but are not limited to,competitive immunoassays, sandwich immunoassays and nucleic acid probeassays. Thus, in embodiments where a reagent is dried on a devicesurface and another reagent is dried on a separate surface of thedevice, these reagents can diffuse from their respective surfaces uponintroduction of fluid to those surfaces. The surfaces having reagentimmobilized thereon can be surfaces in a particular chamber of thedevice or can be surfaces in different regions of the device. Theregions can be separate chambers or can be device surfaces that do notdelimit a chamber.

Additionally, immunoassay reagents can be immobilized on particles ornanoparticles (collectively referred to herein as particles). Suchparticles can be placed on a surface, such as a surface delimiting acapillary space, in a device in accordance with the invention. By use ofsuch particles comprising reagents immobilized thereon, one can providea zone, comprising particles and a surface, where the zone comprisesreagents that could not otherwise be provided together. For example,particles comprising immobilized reagents can be placed on a surfacewhere the surface itself comprises a reagent; when the surface is asurface of a capillary space, one or more capillary space surfaces canhave a reagent immobilized thereon. Different reagents can be placed ondifferent surfaces. A reagent immobilized by the particles (or on asurface) can be diffusible or non-diffusible when placed in contact witha liquid.

Accordingly, use of a device with the preferred configuration hasallowed the performance of one-step immunoassays that simultaneouslymeasure multiple analytes from a biological fluid in an assay time ofabout 10 minutes.

Closing

Although the foregoing invention has been described in some detail byway of illustration and example, it will be obvious that certain changesor modifications may be practiced within the scope of the appendedclaims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aformulation” includes mixtures of different formulations and referenceto “the method of treatment” includes reference to equivalent steps andmethods known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar to equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference to describe and disclose specificinformation for which the reference was cited in connection with.

1. A method for regulating fluid flow in a device that conducts fluidthrough one or more capillary channels, comprising: introducing fluidinto said device which comprises a capillary channel comprising (i) afirst capillary region comprising a hydrophilic surface and(ii) a secondcapillary region comprising a hydrophobic surface adjacent to said firstcapillary region, and a third capillary region comprising a hydrophilicsurface adjacent to said second capillary region, wherein saidhydrophobic surface controls the rate of flow of said fluid into saidthird capillary region, whereby upon introduction of said fluid to saiddevice, fluid flows through said first capillary region to contact saidhydrophobic surface which delays fluid flow into said third capillaryregion until rendered hydrophilic.
 2. The method of claim 1, whereinsaid device comprises a plurality of capillary channels, one or more ofwhich comprise a region comprising a hydrophobic surface.
 3. The methodof claim 1, wherein said device further comprises a vent.
 4. A methodfor regulating fluid flow in a device that conducts fluid through one ormore capillary channels, comprising: contacting said fluid with one ormore hydrophobic regions on a capillary surface that alter a rate ordirection of said fluid flow within said device in comparison to a rateor direction of fluid flow within said device in the absence of saidhydrophobic region, wherein said hydrophobic region retards fluid flowinto a hydrophilic region until said hydrophobic region is renderedhydrophilic.
 5. The method of claim 4, further comprising contactingsaid fluid with a first capillary region and a second capillary regionadjacent to said first capillary region, wherein a difference incapillarity of said first capillary region compared to said secondcapillary region alters a rate or direction of said fluid flow withinsaid device in comparison to the rate or direction of said fluid flowwithin said device in the absence of said difference in capillarity. 6.The method of claim 4, further comprising contacting said fluid with areagent dried on a surface of the device, whereby said reagent dissolvesinto said fluid, thereby lowering the surface tension of said fluid. 7.The method of claim 4, wherein said device comprises a plurality ofcapillary channels.
 8. The method of claim 4, wherein one or more ofsaid hydrophobic regions are flanked by hydrophilic regions.
 9. Themethod of claim 4, wherein at least one of said hydrophobic regionsalter the rate of flow within said device.
 10. A device that conductsfluid through one or more capillary channels, comprising: a capillarychannel comprising (i) a first capillary region comprising a hydrophilicsurface and (ii) a second capillary region comprising a hydrophobicsurface adjacent to said first capillary region and a third capillaryregion comprising a hydrophilic surface adjacent to said secondcapillary region, wherein said hydrophobic surface controls thedirection of flow of said fluid into said third capillary region,wherein said device is configured and arranged such that uponintroduction of said fluid to said device, fluid flows through saidfirst capillary region to contact said hydrophobic surface.
 11. Thedevice of claim 10, further comprising a reagent dried on a surface ofthe device that, when dissolved dissolves into fluid within said device,lowers the surface tension of said fluid.
 12. The device of claim 10,wherein said device comprises a plurality of capillary channels.